Skip to main content

Full text of "Adventures in radioisotope research;"

See other formats


ADVENTURES IN 
RADIOISOTOPE RESEARCH 



ADVENTURES IN 
RADIOISOTOPE RESEARCH 



The Collected Papers of 

GEORGE HEYESY 

in Two Volumes 



Volume One 




PERGAMON PRESS 

NEW YORK . OXFORD • LONDON • PARIS 

1962 



PERGAMON PRESS INC. 

122 East SSth Street, New York 22, N.Y. 

1404 Neie York Avenue N. W., Washington, S D.C. 

PERGAMON PRESS LTD. 

Headington Hill Hall, Oxford 

4 & 5 Fitxroy Square, London W . 1, 

PERGAMON PRESS S.A.R.L. 
24 Rue des Scales, Pari$ V 

PERGAMON PRESS G.m. b.H. 
Kaiserstrasse 75, Frankfurt am Main 



Copyright 

© 

1962 

Pergamoo Press Ltd. 



Library of Congress Card No. 60—12557 



Printed in Hungary by 
The Printing House of the Hungarian Academy of Sciences 




CONTENTS 



INORGANIC AND PHYSICAL CHEMISTRY 

Volume One 
Analytical Applications 

1 The Solubility of Load Sulphide and Load Chromate (with F. Panoth) 31 

2 Platinum Black (with M. Soniya) 36 

3 Lead Content of Rocks (with R. Hobbi) 43 

Activation Analysis 

4 The Action of Neutrons on the Rare Earth Elements (with H. Levi) .... 47 

5 Artificial Activity of Hafnium and Some Other Elements (with H. Levi) ()3 

Electrochemistry 

6 The Problem of the Isotopic Elements (with F. Paneth) 75 



Interchange Studies 

7 The Velocity of Dissolution of Molecular Layers (with E. Rona) <S0 

8 The Exchange of Atoms Between Solid and Liquid Phases 9 7 



9 Intermolecular Exchange of Atoms of the Same Kind (with L. Zech- 

meister 103 

Selfdiffusion 

10 Setf-diffusion in Solid Lead (with J. Groh) 110 

11 Self-diffusion in Solid Metals (with A. Obrutsheva) 114 

12 The Heat of Relaxation of the Lead Lattice (with W. Seith and L. Keil) lir> 

13 Diffusion in Metals (with W. Seith) 122 

14 Apphcation of Radioactive Recoil in Diffusion Measurements (with W. 

Seith) 127 

Tracers In The Search For Unknown Stable Elements 

15 Search for an Inactive Isotope of the Element 84 (Polonium) (with 

A. Gunther) 140 



81310 



6 ADVENTURES IX RADIOISOIOPE ItESEARCH 

LIFE SCIENCE 

Application of Radioactive Tracers Occurring in Nature 

16 Radiochemical Method of Studying the Circulation of Bismuth in 

the Body (with J. A. Chi'istiansen and S. Lomholt) 143 

17 Radiochemical Method of Studying the Circulation of Lead in the 

Body (with J. A. Christiansen and S. Lomholt) 145 

Skeleton Studies 

18 Radioactive Indicators in the Study of Phosphorus Metabolism in 

Rats (with O. Chiewitz) 149 

19 Studies on the Metabolism of Phosphorus in Animals (with O. Chiewitz) 152 

20 Investigations on the Exchange of Phosphorus in Teeth Using Radioactive 

Phosphorus as Indicator (with J. J. Hoist and A. Krogh) 168 

21 Rate of Rejuvenation of the Skeleton (with H. Levi and O. Rebbe) 191 

22 Retention of Atom of Maternal Origin in the Adult White Rat 198 

23 Rate of Renewal of the Fish Skeleton 204 

24 Conservation of Skeletal Calcium Atoms Thiough Life 217 

25 Path of Atoms Through Generations 234 

26 Note on the Chloride Content of the Mineral Constituents of the 

Skeleton 241 



Phosphatides 

27 The Formation of Phosphatides in the Brain Tissue of Adult Animals 

(with L. Hahn) 246 

28 LecithenaemiaFollowingthe Administiation of Fat (withE. Lundsgaard) 255 

29 Formation of Phosphatides in Liver Perfusion Experiments (with 

L. Halm) 258 

30 Rate of Penetration of Phosphatides Through the Capillary Wall 

(with L. Hahn) 262 

31 Origin of Phosphorus Compounds in Hen's Eggs (with L. Halm) .... 273 

32 The Origin of the Phosphorus Compounds in the Embryo of the 

Chicken (with 11. Levi and O. Rebbe) 293 

33 Formation of Milk (with A. H. W. Aten) 304 

34 Formation of Lecithin, Cephalin and Sphingomyehn (with L. Hahn) 309 

35 Turnover of Phosphatides (with G. Ehott) 346 



CONTENTS 7 

Acid Soluble Phosphorus Compounds 

30 Molecular lleju\'cnation oi" Alusclo Tissue (with O. lie b be) 3(j(i 

37 Rate of Renewal of the Acid Soluble* Phosphorus Compounds of the 

Rabbit (with L. Hahn) 3(j9 

38 Ciieulation of Phosphorus in the Fiog (with L. TTahn and O. Rebbo) 384 

Fatty Acids 

39 Turnover Rate of the Fatty Acids of the Liver (with R. Ruyssen and 

L. M. Beeckmans) 403 

40 Effects of Dinitro-Cyclo-Pentylphenol on the Incorporation of Labelled 

Acetate Carbon (^^C) Into Tissue Fractions (with L. M. Beeckmans 
and H. Casier) 408 

41 Determination of the Rate of Renewal From the Rate of Disappearance 

of Labelled ^Molecules 417 

Permeability Studies 

42 Rate of Penetration of Ions Through the Capillary Wall (with L. Hahn) 42o 

4 3 Rate of Passage of Water Through Capillary and Cell Walls (with 

C. F. Jacobsen) 437 

44 Rate of Penetration of Phosphate into Muscle Cells (with O. Rebbe) 443 

4o The Effect of Excitation on Neive Permeability (with H. Euler and 

U. Euler) 454 

46 Note on the Inorganic Phosphate of Blood Plasma (with G. Elliot 

and L. Hahn) 460 

47 Fate of the Sulphate Radical in the Animal Body (with A. H. W. Aten) 465 

48 Diplogen and Fish (with E. Hofer) 468 

49 Interaction of Plasma Phosphate with the Phosphorus Compounds 

Present In The Corpuscles (with A. H. W. Aten) 471 

50 Rate of Penetration of Ions Into Erythrocytes (with L. Hahn) 493 

Volume T wo 
Labelling of Red Corpuscles 

51 A Method of Blood Volume Determination (with L. Hahn) 517 

52 Determination of the Red Corpuscle Content (with K. Zerahn) 523 

53 Thorium B Labelled Red Corpuscles 53 1 

Clinical Investigations 

54 Elimination of Water from the Human Body (with E. Hofer) 536 

55 Excretion of Phosphorus (with L. Hahn and O. Rebbe) 540 



8 ADVEXTUKES IIv" RADIOISOTOPE KESEAECH 



56 Potassium Inteirhange in the Human Body 553 

57 The Red Corpuscle Content of the Circulating Blood Determined by 

Labelling the Erythrocytes with Radio-Phosphorus (with K. H. 
Koster, G. S0rensenj E. Warbvirg and K. Zerahn) 561 

58 Application of ^^K Labelled Red Corpuscles in Blood Volume Measure- 

ments (with G. Nyhn) 573 

59 Application of "Thorium B" Labelled Red Corpuscles in Blood Volume 

Studies (with G. Nylin) 580 

60 Cancer Anaemia 597 



Iron Metabolism 

61 Effect of Adrenaline on the Interaction Between Plasma and Tissue 

Constituents (with G. Dal Santo) 610 

62 Effect of Irradiation on Hemin Formation (with R. Bonnichsen) .... 624 

63 Haemoglobin Present in the Nuclear Fraction of the Liver (with R. 

Bonnichsen, G. Ehrenstein and J. Schliack) 034 

64 Apphcation of Isotopic Indicators in Haematolog\- 639 

65 Note on the Determination of Radioiron (with K. Agner and R. Bonnich- 

sen) 651 

66 Embryonal Iron Turnover (with G. \-. Ehrenstein) 655 



Nucleic Acids 

67 Rate of Formation of Nucdeic Acid in the Organs of the Rat (with 

J. Ottescn) 003 

68 Rate of Renewal of Ribo- and Desoxyribo Nucleic Acids (with E. Ham- 

marsten) 073 

69 Turnover of Ribosenucleic Acid in the Jensen-Sarcoma of the Rat 

(with H. Euler and W. Solodkowska) 080 

70 Life-Cycle of the Red Corpuscles of the Hen (with J. Ottesen) 088 



Studies in Radiation Biology 

71 Effect of X-rays on Nvicleic Acid Formation in the Jensen- Sarcoma 

(with H. Euler) 092 

72 The Effect of X-rays on Nucleic Acid Formation in the Organs 

Of The Rat (with L. Ahlstrom and H. Euler) 721 

73 Turnover of Nucleic Acid in Retrogade Sarcomata (with L. Ahlstrom 

and H. Euler) "-^1 

74 The Indirect Effect of X-rays on the Jensen-Sarcoma (with L. Ahlstrom 

and H. Eulor) ."^ '^44 



CONTENTS 9 

75 Attempts to Find Products Blocking Nucleic Acid Foimation in the 

Circulation of an Irradiated Organism (with L. Ahlstrom, H. Euler 
and K. Zerahn) 7oH 

76 Fate of The Nucleic Acid Inlroduccd into the Circulation (with L. 

Ahlstrom and H. Euler) 7() I 

77 Formation of Nucleic Acid in Sarcoma Slices, (with L. Ahlstrom and 

H. Eulorl 770 

78 Application of Labelled Substrates in the Study of Enzymic Processes 

(with L. Ahlstrom and H. Euler) 78:i 

79 Effect of X-Rays on the Incorporation of Carbon- J 4 into Desoxy- 

ribonucleic Acid ~!) 1 

80 Effect of X-Rays on the Incorporation of Carbon- 14 into Animal 

Tissue 79:i 

81 Effect of X-Rays on the Incorporation of 14-C into Tissue Fractions 

of the Mouse (with G. Dreyfus) 795 

82 Effect of Muscular Exercise and of Urethane Administration on the 

Incorporation of Carbon- 14 into Animal Tissue 82 1 

83 Effect of Irradiation by X-Rays on the Exhalation of Carbon Dioxide 

by the Mouse (with A. Forssberg) 825 

84 Effect of X-Rays and Hormones on Resorption Rate of Injected 

Nai^HCOg, (with A. Forssberg) 828 

85 Note on the Effect of X-Rays and Hormones on the Resoiption Rate 

of Injected Na^HCOg (with A. Forssberg) 8:iS 

86 Effect of Irradiation with. X-Rays on the Catabolism of Methyl- 

alcohol in the Mouse 841 

87 Effect of Irradiation with X-Rays on the Catabolism of Ethylalcohol 

in the Mouse 847 

88 Radioactive Tracers in Radiobiological Studies. The Thiity-Sixth 

Silvanus Thompson Memorial Lecture 851 

Botanical Studies 

89 The Absorption and Translocation of Lead by Plants 87(i 

90 Atomic Dynamics of Plant Growth (with K. Lindersti0m-Lang and 

C. Olsen) 884 

91 Exchange of Phosphorus Atoms in Plants and Seeds (with K. Linder- 

strem-Lang and C. Olsen) 887 

92 Interaction Between the Phosphorus Atoms of the Wheat Seedling 

and the Nutrient Solution 89 I 

93 Exchange of Nitrogen Atoms in the Leaves of the Sunflower (with 

K. Linderstrem-Lang, A. S. Keston and C Olsen) 905 

94 Zinc Uptake by Neurospora (with I. Andersson-Kotto) 910 



10 ADVENTURES IX EADIOISOTOPE RESEARCH 

95 Phosphorus Exchange in Yeast (with K. Linderstrom-Lang and N. Niel- 

sen) 9 IG 

96 Potassium Interchange in Yeast Cells (with N. Nielsen) 918 

97 Note on the Number of Pollen Grains Identified in the Fruit of the 

Aspen (with C. Eklundh-Ehrenberg and H. Euler) 924 



Lectures 

98 Some Applications of Isotopic Indicators. Nobel Lecture 928 

99 The Application of Radioactive Indicators in Biochemistry. Faraday 

Lecture 961 

100 Historical Progiess of the Isotopic Methodology and its Influences 
on the Biological Sciences. Read at the Turin Meeting of the 
Society of Nuclear Medicine 997 

Index 1039 



First communicated in Perspectives in Biology aud Medicine 
Vol. I, No. 4, Summer 1C58 

A SCIENTIFIC CAREER 

George Hevesy, 

Ph.D. (hon.), Ph.nat.D. (lion.), D. Sc. (hon.), Sc. D. (hon.) M. D. (lion.) 

Jur. D. (hon.). 

I WAS born in Budapest the 1^* of August, 1885. After terminating 
my studies at the Gymnasium of the Piarist Order in that city, I studied 
a short time in Budapest and Berlin and later in Freiburg, mainly che- 
mistry and physics, where I took my degree in 1908 an. The subject 
of my doctoral thesis was the interaction between metallic sodium and 
molten sodium hydroxide, an interaction responsible for a poor yield 
often obtained when producing sodium by electrolysis of molten sodi- 
um hydroxide. 

Being interested in high-temperature chemistry, I proceeded to Zurich 
after obtaining my degree to work under Richard Lorenz, at that 
time the most eminent representative of that branch of science. The 
Technische Hochschule of Ziirich was in those days, as it is today, a 
great place of learning and teaching. The Swiss chemical and pharma- 
ceutical industry could not have reached its present high standard it 
represents today without the aid of a great number of able chemists, 
most of them trained at the Technische Hochschule of Ziirich. When 
I joined this institution, the permanent head of the chemistry depart- 
ment was Willstatter. 

Einstein's First Lecture 

Shortly after my arrival at Ziirich, Einstein was appointed associate 
professor of theoretical physics on the University. I was one of the 
audience of about twenty who attended his inaugural lecture on the 
determination of the ratio of charge and mass of the electron. (Einstein 
left after a few years for Prague and returned later to Ziirich to fill the 
chair of theoretical physics on the Technische Hochschule.) When he 
visited our laboratory,! had the privilege to show him around. I remember 
vividly his astonishment w^hen shown a hydrogen electrode. He thought 
such an electrode to be only a theoretical concept. 

Twenty-three years later, after terminating my Baker lectureship on 
the Cornell University at Ithaca, I met Einstein in Pasadena. I visited a 



12 ADVENTURES IX RADIOISOTOPE RESEARCH 

barber shop whose owner, a son of the City of Constance, praised the 
beauties of Ufe in California, mentioning that his only wash in life was 
to be permitted once to cut Einstein's hair. I told him that this wish 
would not be easy to fulfill as, according to rumors, Mrs. Einstein 
used to perform this work. When I told Einstein about the barber's 
w ish, he remarked : ''Da er sich auf Ihrem Kopfe nicht austoben konnte, 
wollte er meinen Kopf haben" ("As he could not sufficiently exercise 
himself on your head [I had poor hair] he wants to have mine"). 

Einstein talked repeatedly to me on the problem of causality. He dis- 
agreed with Bohr's views on this topic and asked me to convey his 
objections to Bohr. He wished an explanation on a classical basis. 

When Lorenz left Ziirich for the University of Frankfurt (I was 
later, after his death, asked to fill his chair). Willstatter called on 
me to make the short statement : ''In Germany the assistant belongs 
to the professor, in Switzerland to the laboratory— you stay here." 
I did not, as I got much interested in the catalytic synthesis of ammonia 
by Haber, a discovery which at that date rightly impressed all those 
interested in chemistry. 

My monthly salary in Ziirich corresponding to $36 was entirely ade- 
quate, as I was charged $15 a month for a very nice room and two good 
meals a day. When I was promoted to a "first assistant", I was told that 
my salary would be raised to $60 a month, the highest pay ever allotted 
to an assistant. 

When I w-as leaving the laboratory one evening together with Will- 
statter, he told me that he was moving to Berlin to take over one of the 
Kaiser Wilhelm Institutes. I asked him, much astonished, w^hy he was 
leaving. He was the permanent head of the chemistry faculty and had 
a very fine laboratory, and postgraduate students from all over the world 
were anxious to work under his guidance. His answer was : "If the 
fatherland calls, it is my duty to go." Thirty-two years later I was present 
at the meeting of the Danish Academy of Sciences when the president, 
S. P. L. Sorensen, death written on his face, read a letter from Willstatter 
requesting that the Proceedings of the Academy should no more be sent 
to him. Willstatter went on, saying : "I have no home any more. I have 
lost all my belongings, which I do not mind much. What chagrins me is 
that I lost my fatherland." 

Haber wished me to work in another field than that of catalytic synthe- 
sis. I was to investigate whether or not oxidation of molten zinc is 
accompanied by emission of electrons. No one in Haber's institute had 
experience in the field of the conductivity of electricity in gases. I pro- 
posed therefore to Haber that I proceed to England to acquire some 
knowledge in this new field of physics and return later to his laboratory. 
Haber entirely shared my view, and I left in the first days of January , 
1911, for Manchester to work under Rutherford. 



A SCIENTIFIC CAREER 13 

Years with Rutherford 

The physics laboratory of the University of Manchester was housed in 
a spacious building. The chief equipment of the institute was electro- 
scopes built with cocoa cans, sealing wax, sulphur rods, gold leaves, and 
reading microscopes. Once adjusted, the electroscopes were not permitted 
to be cleaned, and the smokey atmosphere of Manchester left its visible 
marks all over the laboratory. The years I had the privilege to spend 
in Rutherford's laboratory in Manchester, between 1911 and 1914, 
witnessed some of the greatest discoveries in the history of physics. 
I could follow from close quarters the discovery of the atomic nucleus and 
how Rutherford devised, carried out, and interpreted the results of ex- 
periments. All this was done with the greatest ease, without visible effort. 

Niels Bohr came to Manchester in 1912. He recently remarked in 
an after-dinner speech that I was the first-one he met when he entered 
Rutherford's institute. Rutherford— and not he alone— soon realized 
Bohr's genius. When I was enjoying Rutherford's hospitality one Sun- 
day afternoon, soon after the discovery of the atomic nucleus, I happened 
to ask him about the origin of/3-rays. Thea-rays clearly originated from 
the nucleus, but what about the origin of /9-rays ? Rutherford answered 
promptly, "Ask Bohr", and the answer was at once given by the latter, 
emphasizing the difference between nuclear and non-nuclear ^-particles. 
Bohr was however not always easy to understand. When he briefly stated, 
"Argon is not the right argon", he made a statement that was at that 
date not easy to interpret. It was then, in 1912, already clear to him 
that it is not the mass number but the atomic number that is decisive 
for the place of an element in the periodic system. Soon after, this fact 
was decisively brought out by Moseley's work. I consider myself lucky 
to have had the opportunity to help Moseley set up the first X-ray 
spectrograph. We turned to the steward of the chemistry department, 
Mr. Edwards, who handed us a beautiful, very large potassium ferri- 
cyanide crystal which found application in Moseley's spectrograph. A mag- 
netic device served to bring small metal disks covered with the element 
to be investigated into the electron beam, which had to excite the X-rays. 
Moseley's fundamental work brought out, among other things, that, 
while the atomic weight of argon is higher than that of potassium, its 
atomic number is not— that "argon is not the right argon", as stated by 
Bohr previously. Moseley has also shown that the anomaly of the posi- 
tions of tellurium and iodine in the periodic system disappears if we 
consider the atomic number instead of the atomic weight. 

When I arrived at Manchester, Rutherford wished me to study the 
solubility of actinium emanation in various liquids. It was not an easy 
task in view of the short life of this emanation, now called actinon, the 
half-life of which is four seconds only. This was, however, a very good 



14 ADVENTURES IN RADIOISOTOPE RESEARCH 

school to learn the handling of short-lived substances. I later became 
engaged with the study of the electrochemical properties of radioele- 
ments of unknown chemical character and the measurement of their 
valency from diffusion data. 

The early origin of the famous Geiger- counter goes back to those 
Manchester days as well. Rutherford and Geiger counted a-particles by 
making use of a galvanometer which registered the arrival of each 
a-particle. The ionization produced was magnified by using the principle 
of production of ions by collision. The much more difficult task of 
counting /3-particles was solved later, after the first World War, by 
Geiger, then at Kiel. 

When I was in Manchester, Rutherford was much interested to come 
into the possession of a strong radium D sample. Large amounts of 
radium D were stored in the laboratory, but imbedded in huge amounts 
of lead. The great German chemist Haber intended to pay Germany's 
war debts after the first World War by extracting gold from the ocean. 
First he undertook to check the correctness of the available gold analyses 
of sea water. He found the gold content of the ocean to be very much 
lower than previously found. He summarized the depressing results 
of his expedition by stating : "Dilution is the death of all value". Ruther- 
ford could have made the same remark when glancing at the hundreds 
of kilograms of lead chloride extracted from pitchblende and presented 
to him by the owner of the Joachimsthal mines, the Austrian govern- 
ment. 

Radioactive Tracers 

One day I met Rutherford in the basement of the laboratory where 
the lead chloride was stored. He addressed me by saying : "If you are 
worth your salt, you separate radium D from all that nuisance of lead." 
Being a young man, I was an optimist and felt sure that I should succeed 
in my task. Trying during a year all sorts of separation methods and 
making the greatest efforts, it looked sometimes as if I succeeded, but 
I soon found out that it was radium E, the disintegration product of 
radium D, a bismuth isotope, which I separated. The result of my efforts 
was entire failure. To make the best of this depressing situation, I thought 
to avail myself of the fact that radium D is inseparable from lead, and 
to label small amounts of lead by addition of radium D of known acti- 
vity obtained from tubes in which radium emanation decayed. From 
such tubes pure radium D can be obtained. 

It was the Vienna Institute for Radium Research which owned in 
those days by far the greatest amount of radium and, correspondingly, 
of radium emanation. This fact induced me to interrupt my stay in 
Manchester and to proceed to Vienna. In the Vienna Institute there were 



A SCIENTIFIC CAREER 15 

very large amounts ol lead chloride, oljtained liom pitchblende as well, 
and Paneth, assistant at the Institute, unaware of my efforrs at Man- 
chester, made very extensive studies to achieve separation. His great 
efforts were as abortive as mine. At my suggestion we associated in the 
application of labelled lead. The first use of this method, early in 1913, 
was the determination of the solubility in water of sparingly soluble 
salts such as lead sulphide and lead chromate. We then proceeded to 
study the electrochemistry of bismuth and lead by making use of the 
method of radioactive indicators. We could show, among other things, 
that Nernst's law of the dependence of the electrode potential on the 
ionic concentration is valid even at exceedingly low concentrations. 
Paneth then directed his interest toward the interaction of the lead ions 
present in the surface layer of lead sulphate and the labelled lead ions 
of the surrounding solution. I studied the interaction of the lead atoms 
of a lead foil and also of lead peroxide with the lead ions of a solution, 
employing labelled lead foils and non-radioactive lead salt solution, or 
vice versa. In the last of the numerous joint investigations with Paneth, 
we succeeded in preparing visible amounts of radium D from radium 
emanation. By comparing the electrode potential of radium D peroxide 
with that of lead peroxide, we were able to show that these cannot be 
distinguished from each other. 

During my stay in Vienna, I undertook balloon ascents in the company 
of Hess and Paneth. On one of his trips Hess took an electrometer with 
him to follow the change in the ionization of the air with height. He 
assumed this ionization to be due to terrestrial radiation and corres- 
pondingly expected it to decrease with height. The opposite, however, 
was found to be the case. With such simple means and without much 
effort this observation led to the discovery of cosmic radiation. 

Madame Marie Curie 

When passing through Paris on the w^ay to Manchester, I never failed 
to call on Marie Curie and I was always sure to find her amidst experi- 
mental work. She was usually surrounded by several girl assistants 
precipitating or crystallizing preparations. The only protection that she 
used was finger caps of rubber. When engaged with the concentration 
of actinium from rare-earth samples, she generously presented me with 
an actinium preparation. I consider this specimen one of my most pre- 
cious belongings. As the years pass by, the bottle containing the radio- 
active sample is getting more and more coloured, indicating the many 
years which have elapsed since I met this most remarkable personality 
and great pioneer. 

At a later visit to the Institut de Radium, I met Joliot, who was then 
a young assistant engaged in the study of the electrochemistry of polo- 



\ Q ADVENTURES IN RADIOISOTOPE RESEARCH 

nium, which many years earher was in the center of interest of Paneth 
and myself. Also, Irene Curie worked in the laboratory of her mother. 
WhenI saw her in 1938, she mentioned that by neutron bombardment 
of thorium she had obtained a lanthanum-like radioactive body. I asked 
her if she was sure that this substance was not actinium. She answered 
that she was pretty sure she was dealing with an element much lighter 
than one of the radioactive disintegration series. 

A few months later Otto Hahn and Strassman made their fundamental 
discovery of nuclear fission. I first met Hahn in Vienna in 1913. Already 
at that date he had made such important discoveries as the existence 
of radiothorium and mesothorium and the separation of radioelements 
by making use of the recoil phenomenon. The years to come, brought 
new discoveries of great importance, many of them in collaboration with 
Lise Meitner. When I asked Rutherford in 1912 whom of his students 
he considered to be the most merited one, he answered without hesitation 

"Otto Hahn". 

On my way to Manchester I usually stopped in London. On such an 
occasion I had the opportunity of being present in the House of Commons 
at the introduction of the much discussed budget by Lloyd George, then 
Chancellor of the Exchequer, who characterized his introduction of heavy 
death duties and other taxes as "bringing rare and refreshing fruit"! 

I was also present when J. J. Thomson in April, 1913, delivered his 
Bakerian Lecture in the Royal Society on the two neon parabolas ob- 
tained in his positive ray studies. He did not make any allusion to the 
analogy between the two neons and the isotopes in the field of radioacti- 
vity. This omission induced me to write to him drawing his attention 
to the analogy between the two kinds of neon, on one hand, and radium 
D and lead, on the other. He stated in his answer that he did not share 
my view. While not adopting the view that the heavier constituent of 
neon was a compound NeHg, which could have given the observed ato- 
mic weight within the limits of experimental error, Thomson was not 
convinced that this explanation was absolutely excluded. As Lord Ray- 
leigh remarks in The Life of Sir J. J. Thomson, he had always been 
haunted by this suspicion about hydrogen compounds and, for that rea- 
son, hesitated for a time to accept Aston's later results about isotopes 
of other elements. When we were on a ski-trip at Finse in Norway, Aston 
related that when he first succeeded in getting two lines on a mass 
spectrum photograph - one indicating ^^C\, the other ^^C\ - Thomson 
refused to look at the photograph, which, Aston added, was the most 
beautiful one he ever obtained. Aston was an ingenious and most merited 
experimenter, who was the first one to prove the complexity of the com- 
mon elements. 

In 1914 Moseley moved to Oxford and, being much interested in 
X-ray spectroscopy, 1 intended to work with him. We wanted to study 



A SCIENTIFIC CAKEEll 17 

the X-ray spectrum of the elements 68 through 12. 1 was ah-eady in 
Holland on the way to Oxford when the first World War broke out, soon 
followed by the tragic death of Moseley. While talking on a field telephone 
at Gallipoli, a bullet struck the head of this ingenious and most remarkable 
man. By a curious coincidence, I was to occupy myself extensively with 
the X-ray spectrum of the element 72 eight years later. 

Measurement of Self -diffusion 

As I was at that time a Hungarian subject (1 am now a Swedish citizen) 
1 was drafted into the Austro — Hungarian army. I spent much of 
1hat time as technical supervisor of electrolytic copper works. While 
located in Carpathian plants, I fitted up a laboratory on a very modest 
scale and studied the difference in the chemical behaviour of the active 
deposit of thorium when present in ionic and colloidal state. For several 
months after the end of the war it was not possible to leave Hungary. 
During these months I started with my friend Groh to study self-diffusion 
in molten and in solid lead, using radium D as an indicator. We fused 
a radiolead-rod on to the top of an inactive lead-rod, heated this solid 
system to 200° — 300°, and determined the dislocation of the radium D 
atoms. From the extent of dislocation, the rate of self-diffusion of lead 
was calculated. This early, rough method was improved later during 
my stay in Copenhagen. Together with the Russian scientist Mrs. Ob- 
rut she va, we condensed the lead isotope thorium B on top of a lead foil 
and counted the number of scintillations produced by the a-rays emitted 
by the disintegration products of thorium B. Upon heating of the sample, 
thorium B diffused into the lead foil, resulting in a reduction of the 
number of scintillations observed. From this reduction, the diffusion 
rate of lead in lead could be calculated. Heisenberg, then lecturer in 
Copenhagen, very kindly at that time helped us with these calculations. 
Later on in Freiburg, Seith and myself made use of the recoil pheno- 
menon to measure self-diffusion in lead. This is an exceedingly sensitive 
method, which permitted measurement of diffusion rates as slow as 
10-14 cm2/day. 

The Rockefeller Foundation started to support my investigations in 
1930 and continued most generously to do so for the following twenty- 
five years. 

Niels Bohr's Institute 

In the first days of May, 1919, I left for Copenhagen to spend some 
time with Niels Bohr at the charming summer house in Tibirke. At that 
time his premises were at the Technological Institute of Copenhagen, 
from which he directed the construction of his new institute. When he 

2 Heresy 



18 ADVENTURES IN RADIOISOTOPE RESEARCH 

had to decide on a name for the new institute, he hesitated between 
"Theoretical Physics" and "Atomic Physics". His choice fell on the first 
one ; he felt that the latter might be too exacting and possibly too special 
as well. In front of the Technological Institute there is a statue of Olaus 
Romer, the first physicist to measure the velocity of light. I once pointed 
out when passing this monument that space is available for a future 
monument of Niels Bohr. My companion smiled at this remark. Today 
he would not smile any more. 

It was settled with Bohr that I should be back in Copenhagen in the 
spring 1920, to start activities at the new institute which was to be opened 
by that date. I spent the remaining six months with my friend Zech- 
meister in Budapest carrying out exchange studies by the application 
of radioactive indicators. When dissolving in water both 1 mol of labelled 
lead nitrate and 1 mol of non-radioactive lead chloride, or labelled lead 
chloride and non-radioactive lead nitrate, after separation of the two 
compounds, we found the radioactivity equally distributed between 
PbCla and PbNOg. When dissolving non-radioactive tetraphenyl lead 
and radioactive lead nitrate, after separation all radioactivity was con- 
served in the nitrate sample, as the lead atoms of tetraphenyl lead are 
not exchangeable. When I met Svante Arrhenius in 1922 he told me 
about his interest in the above-mentioned work. The experiments with 
labelled lead chloride and non-labelled lead nitrate, or vice versa, are 
the most direct proof of the correctness of the theory of electrolytic 
dissociation. 

After the war, I was anxious to go to England as soon as possible. The 
atmosphere at that time, however, radically differed from the one that 
prevailed after the second World War. When in 1921 I wrote from Copen- 
hagen to Rutherford, a very liberal man, that I wished to visit England, 
he answered that it was still too early for a former enemy to come to 
England. In 1923, however, when he was elected president of the British 
Association meeting which was to take place in Liverpool, he invited 
me to address that meeting on the discovery of hafnium. I recall a lunch 
party at Liverpool in which Lord and Lady Rutherford, Niels Bohr, 
Millikan, Aston, Coster, and myself took part. Lady Rutherford remarked 
that this party included four Nobel Laureates. Rutherford added, 
"And some embryos". 

Rudolf Schoe nheimer 

During my stay in Liverpool I was told about the work of Blair- 
Bell who claimed a successful cancer therapy through administration 
of lead compounds. These results induced me, when I worked at the 
University of Freiburg some time later, to study the distribution of 
labelled lead compounds between cancerous and normal tissue. A study 



A SCIENTIFIC CAKEEK 1 9 

of the distribution of labelled lead and bismuth in healthy rabbits was 
carried out earlier, in 1924, in Copenhagen. I approached the great 
pathologist Aschoff to delegate one of his collaborators to help us in 
our work. He first delegated the director of a hospital on the island of 
Formosa and later, to help him, his chief chemist, Rudolf Schoenheimer. 
This was Schoenheimer's first experience with tracer work, a field to 
which he later, jointly with his eminent colleague Rittenberg, made 
unsurpassed contributions. Schoenheimer was already at that date a 
very nervous man. He moved his limbs incessantly, smoked cigarettes, 
and consumed coffee on a much too liberal scale. When our work was 
finished, he left Freiburg and I never saw this most merited man again. 

Separation of Isotopes 

When I went to Copenhagen in the spring of 1920, Bohr's institute 
was not yet ready. I associated with the eminent physicochemist Br0n- 
sted to investigate a problem of great interest to both of us, namely, the 
partial separation of isotopes on a preparative scale. We based our pro- 
cedure on the more rapid rate of evaporation of the lighter isotope from 
a liquid. We distilled mercury in high vacuum at 40° and prevented the 
more rapidly evaporating lighter isotopes from being reflected back into 
the liquid mercury by freezing them on a glass surface cooled with 
liquid air. By repeating this process some hundred times, we obtained a 
light and a heavy mercury fraction. The results were controlled by both 
density measurements and atomic weight determinations, the latter 
being carried out by Honigschmid in Munich. 

When partially separating the isotopes of chlorine, we made use of the 
above-mentioned method again. We distilled concentrated solutions 
of hydrochloric acid in water and obtained several liters of water con- 
taining hydrochloric acid with different isotopic chloride composition. 
I suggested to Br0nsted that he have a look at the density of the water ob- 
tained. He objected to my suggestion, as shortly before two distinguished 
German chemists, Vollmer and Stern, had searched without success for 
other isotopes of hydrogen and oxygen than ^H and ^^0. These workers 
carried out diffusion experiments through porous membranes. When I, 
after Urey's discovery of deuterium, reminded Br0nsted of my suggestion, 
he answered : "A discovery like this should not be made fortuitously ; 
it should be based on careful considerations like Urey's." 

Bohr was highly interested in our separation experiments and keenly 
followed our progress. Bohr's greatness is due not only to his ingenuity 
but to the unique catholicity of his interests, his sagacity, and his immense 
conscientiousness. When as a young man he intended to publish his 
first "letter" to the editor of Nature, he wrote the note ov^r and over 
again. Finally his brother, who later achieved fame as a great mathema- 

2* 



20 ADVEXTURES IS RADIOISOTOPE RESEARCH 

tician, suggested he should now mail the "letter", Niels Bohr was shocked 
by this suggestion, since, he said, this was the first trial of the first con- 
cept of the "letter." In this spirit all his papers were written. 

How fabulously far-sighted Bohr was, is seen from a letter which the 
present writer addressed to Rutherford after the Birmingham meeting 
of the British Association for Advancement of Science from Budapest 
the 14th October 1914. 

"The meetings on Monday and Tuesday have been very interesting. 
It is a most remarkable fact that Aston succeeded to separate the two 
Neons by diffusion and gave a definite proof that elements of different 
atomic weights can have the same chemical properties. Thomson came 
in his paper on Xg to the conclusion that the latter is a polymerized 
hydrogen, a kind of H3 (like O3). In the following discussion Bohr— in his 
usual modest way — suggested the possibility that X3 being an H atom 
with one central charge, but having a three-times heavier nucleus than 
hydrogen. He suggested to let a mixture of H and X3 diffuse through 
palladium and try if it is possible to separate them, as the heavier X3 
atom has to diffuse much slower. 

"Bohr had not been understood properly and Thomson gave a rathei 
quick answer, saying — after a brief consultation with Ramsey — 
that Bohr's suggestion is useless, for not molecules, but the atoms 
of H diffuse through Palladium. Certainly, but this was just Bohr's 
point. 

"The general appearance was, that he told something highly ingenious 
and Bohr something very stupid. Just the contrary was the case. So I 
felt bound to stick up for Bohr and explained the meaning of Bohr's, 
suggestion in more concrete terms, saying that Bohr's suggestion is that 
X3 is possibly a chemically non-separable element from Hydrogen . . . 
Of course not very probable, but still a very interesting suggestion; 
which should not be quickly dismissed" ... 26 years later Tritium was 
discovered. 

Simultaneously with the isotope separation studies, I carried out 
among other things some tracer- work on the interchange between the 
atoms of lead compounds and lead, all in molten state. 

In 1921 Bohr's institute was opened. Those working at the institute 
at its start were, besides its director, H. Kramers, H. M. Hansen, I. C. 
Jacobsen, James Franck, who was invited for a short visit, and myself. 
In my first study at the institute I measured the conductivity first 
of a single crystal of sodium nitrate and then after it was molten and 
resolidified into a polycrystalline mass. This crystalline conglomerate 
was found to have a specific conductivity, fifty times higher than the 
single crystal. From this result it was concluded that deviations from the 
ideal crystalline state promote electrolytic conductivity. While increase 
of temperature produces a reversible loosening of the lattice, we are 



A SCIEXTIFIC CAKEEK 21 

here faced with an "irreversible loosening" of the crystal structure. 
This was a most modest beginning in a field tlial later proved to be of 
great importance. 

Hafnium 

Bohr's first fundamental papers, published in 1913, in which the quan- 
tum theory of the atomic structure was introduced, dealt only with the 
structure of the atoms of hydrogen, helium, and lithium. In January, 1922, 
I learned during a walk together with him that he now had extended his 
theory to the entire periodic system, giving among other things an ex- 
planation of the appearance of the rare-earth elements in that system. 
Their number according to his theory was restricted to fourteen, from 
which it followed that the unknown element 72 cannot be a rare earth, 
it has to be a homologue of the titanium group. 

In the summer of that year I became interested in geochemical prob- 
lems. Returning to Denmark, I proposed to Coster, who previously had 
studied X-ray spectroscopy with Siegbahn in Lund, that he should 
teach me the technique and that we ought at the same time to have a 
look at zirconium minerals for the missing element 72. The first spectrum 
obtained by him demonstrated the presence of the element in zirconium 
minerals, and further studies revealed its presence in all commercial 
zirconium samples, which indicated a very close kinship between zirco- 
nium and the new element hafnium. By a very protracted fractional 
crystallization of ammonium zirconium hexafluorides, hafnium could 
be prepared in a pure state. 

The discovery of hafnium was not accepted without opposition. 
Urbain, in Paris, a few years earlier crystallizing crude ytterbium salts, 
observed twenty-six optical spectral lines not shown by the initial 
sample. He ascribed these lines to the presence of the previously un- 
known element 72 in his sample. After the discovery of hafnium, it was, 
however, demonstrated that none of these lines is to be found in the 
spectrum of hafnium. In spite of this fact, Urbain upheld his claim to 
have discovered element 72. Rutherford took great interest in our 
work— all our extensive correspondence with iVa^Mre passed through his 
hands — and suggested that I should send a paper on the chemistry of 
hafnium to the editor of Chemical News. He remarked in his letter that 
the editors of this periodical were strongly pro-French and I should not 
mind if they refused to publish my paper. In Sheffield, my friend the 
physicist Lawson ("interned" as a prisoner of war in the Institute of 
Radium Research of Vienna) handed my contribution over .to the editor 
of Chemical News, Professor Wynne. He remarked that he was pleased 
with the paper but they might have something to say about the name 
"hafnium," adding : "We adhere to the original word Celtium given to 



22 ADVENTURES IN RADIOISOTOPE RESEARCH 

it by Urbain as a representative of the great French nation which was 
loyal to us throughout the war. We do not accept the name which was 
given it by the Danes who only pocketed the spoil after the war." The 
paper was, however, published by Chemical News without remark. 

Another opposition to the discovery of hafnium came from London. 
Alexander Scott, the chief chemist of the British Museum, could not iden- 
tify a fraction of a sample of an Australian titaniferous sand. After our 
discovery was announced, he thought this fraction to be hafnium. 
Scott's paper induced the Times to publish in its February 2, 1923, 
issue an editorial under the title "Hafnium", stating : "Science is, and 
doubtless should be, international, but it is gratifying that this chemical 
achievement, the most important since the late Sir Wilham Ramsay 
isolated helium in 1895, should have been the work of a British chemist 
in a London laboratory." Scott's sample, sent us for investigation, did 
not contain a trace of hafnium or zirconium. 

The determination of the hafnium content of a great number of zir- 
conium minerals and historical zirconium samples was a fascinating 
task. Berzelius determined the atomic weight of zirconium by analyzing 
its sulphate. This method supplies too low values for the atomic weight. 
This error was, however, compensated by the presence of hafnium, almost 
twice as heavy as zirconium, in his sample. Venable in South Carolina, who 
spent many years with the determination of the atomic weight of zir- 
conium, applied a modern method devised by Richards at Harvard. He 
could not find the reason why his determination led to a clearly too high 
value. After the discovery of hafnium, he sent us a sample of his zirco- 
nium, and, after taking into account its quite appreciable hafnium 
content — which we determined— he could correct the presence of 
hafnium in his sample and arrive at a precise value for the atomic 
weight of zirconium. 

Through my work with hafnium I came into contact with the great 
Austrian chemist Auer von Welsbach. He invested a part of his very 
substantial royalties obtained for his patent of cerium-iron alloys (applied 
in cigar-lighters among other things) in a beautiful estate in Carinthia on 
which he built a castle. The rough crystallization of rare earths was 
carried out in one of his nearby situated works, and the final crystalliz- 
ation was done by himself in his castle. He was at that date and for 
many years to come the only man who possessed highly purified samples 
of all elements of the rare-earth group. When staying with him, he 
expressed his astonishment that when separating hafnium from zirco- 
nium I had chosen to handle large amounts of fluorides, which are highly 
unpleasant compounds to work with. He achieved all his great success 
in the field of rare-earth chemistry by crystallizing double- sulfates. We 
found out later that there is no significant difference between the solu- 
bility of zirconium and hafnium double- sulfates, and if we had chosen 



A SCIEXTIFIC CAREER 23 

to crystallize these compounds, we would not have been able to separate 
hafnium from zirconium. All hafnium commercially available for the 
next twenty-five years was prepared by crystallizing the double- fluorides. 

V. M. Goldschmidt 

Auer von Welsbach presented me with small samples of octohydro- 
sulphates of all elements of the rare-earth group. This gift enabled me to 
measure the density of these compounds and to observe a systematic 
decrease of the size of the ions of rare-earth elements when proceeding 
from cerium to lutetium, a contraction which explained the extreme 
kinship of zirconium and hafnium, which are more closely related chemi- 
cally than any other elements of the periodic system. (When testing the 
rare earths for radioactivity, making use of Auer von Welsbach's samples, 
we discovered that samarium emitted a-rays.) In Oslo, V. M. Gold- 
schmidt simultaneously observed the contraction of ionic size, proceed- 
ing from one rare earth to the next one and denoted this rare-earth con- 
traction as the "lanthanide contraction." Goldschmidt described his and 
my work in his posthumously published book Geochemistry , a most 
fascinating reading, like everything that he wrote. V. M. Goldschmidt 
was one of the most able men I ever met. Endowed with an immense 
knowledge and a fabulous memory, he was full of fertile ideas. 

A few weeks prior to the occupation of Norway, I spent a few^ days 
with him at his home on Holmenkollen near Oslo. He predicted the 
tragic happenings of the coming years, which very few foresaw. He 
mentioned that his pupil and former assistant Lunde soon would become 
a "Gauleiter" of Norway. Lunde was later the Minister of propaganda 
in the Quisling government. Goldschmidt predicted that the Norwegian 
coast batteries would fail to fire at the invading enemy, which they in 
fad did with very few exceptions. He was also endowed with much 
humor. When Quisling came into power, Goldschmidt was imprisoned 
and all his property seized. Being short of phosphorus fertilizers, the 
government released him and instructed him to prepare phosphorus from 
Norwegian minerals. All his property, however, remained confiscated. 
When German colleagues passed en route to Rjukan, where they had to 
inspect the heavy- water works, they called on Goldschmidt. He invited 
them for dinner, encouraging them to eat with the remark : "Please go 
on eating, gentleman, all you consume is state property." 

Tracers in Biology 

During the work willi hafnium, I continued the tracer work and in 1928 
applied radium D and thorium B as tracers in the study of the uptake of 
lead by bean seedlings and also in the removal of labelled lead by non- 



24 ADVEXTURES IN RADIOISOTOPE RESEARCH 

labelled lead from such seedlings. This was the first application of radio- 
active tracers in biological studies. The following year we extended 
these studies with my friends Christiansen and Lomholt to the distri- 
bution of lead and bismuth in the animal organism. 

Potassium is one of the few radioactive elements found in nature 
outside the members of the disintegration series. We were interested 
to find out which of the potassium isotopes is radioactive. For this 
purpose we carried out a partial separation of the potassium isotopes, 
applying the same method used when separating the isotopes of mercury. 
A few kilograms of metallic potassium were distilled and a heavy and a 
light potassium fraction obtained. From the difference in the activity 
of these samples and the difference in their atomic weight, the mass 
number of the active isotope could be calculated. Among other instru- 
ments that were used to measure the activities of our samples was the 
first counter built by Geiger in his institute at Kiel. The atomic weight 
of our sample was determined by Honigschmid in Munich. From these 
data it was concluded that the mass number of radioactive potassium 
is 41. 

The first one to draw my attention to the fact that this result was 
probably wrong was Baxter, when I visited him at Harvard. He had 
found that, in constrast to all other atomic weight figures determined 
by Honigschmid, that of potassium was wrong. Baxter proposed to 
determine the atomic weight of our potassium samples. From his results 
it followed that the mass number of radioactive potassium is 40. The 
two greatest authorities in the field of atomic w^eight determination thus 
arrived at different results as to the atomic weight of our potassium 
samples. To reach a decision, we extracted the small calcium content 
of an old potassium-rich mica. If '^iR were the active isotope, then 
the calcium isolated should contain ^-Ca. Aston could not, however, 
find any ^^Ca in our sample. Thus ^^K does not disintegrate and 
is not radio- active. Baxter was right. A few years later Fermi and 
collaborators observed the production of an artificial potassium isotope 
when bombarding potassium with neutrons. We obtained with Miss 
Hilde Levi Fermi's product by bombarding scandium and also calcium 
with neutrons. As scandium has only one stable isotope, we could 
conclude from our investigations that Fermi's radiopotassium has the 
mass number of 42. 



Activation Analysis 

Auer von Welsbach was very cautious in giving away his very valuable 
rare-earth samples, but one day when I was staying with him he was 
in a generous mood and told me to choose one of his samples, of which 



A SCIENTIFIC CAREER 2 



Ziy 



he said he was willing to give me a larger amount. 1 chose dysprosium 
without having any special reason to do so. Ten years later, after the 
discovery of artificial radioactivity, we exposed Auer's dysprosium to 
slow neutrons and succeeded in producing an exceedingly strongly active 
radiodysprosium. No element is known that can be actived more inten- 
sively than dysprosium and europium. Exposure of Auer's europium to a 
neutron beam also led to the formation of a very strongly active radio- 
europium, while no active gadolinium could be prepared by using radium- 
beryllium as the source of neutrons. At that time my friend Professor 
Rolla, of the University of Florence, who prepared a few kilograms of 
gadolinium oxide, sent me samples of this material which he wished us to 
analyze for europium by X-ray spectroscopy. We had earlier analyzed 
several of his samples quantitatively applying secondary X-rays, a 
method which was worked out in the Freiburg laboratory. Having no 
access to a Roentgen spectroscope in Copenhagen at this time, we tried 
together w-ith Miss Hilde Levi to analyze the samples by exposing 
them to a flux of slow neutrons. All the samples contained some euro- 
pium . 

By preparing standards containing a known amount of pure gado- 
linium and pure europium, we could arrive at quantitative figures for 
the europium content of Rolla's samples. This was the start of activa- 
tion analysis, which has since become an important tool in analytical 
chemistry. It was possible by this method to determine, for example,, 
the minute amounts of sodium and potassium in a nerve fiber. 

Deuterium as a Tracer 

Urey's epochal discovery of deuterium took place while I worked in 
Freiburg. Most kindly he promptly supplied us with some liters of 
waters containing 0.6 per cent of deuterium oxide. This low heavy- 
water concentration sufficed to study the interchange of the water 
molecules between goldfish and the surrounding water and also to 
determine the water content of the human body, making use of the 
principle of isotope dilution already introduced a few years earlier 
(1931) when we determined the lead content of rocks. The mean lifetime 
of the w^ater molecules in the human body was determined as well. 
When I returned to Copenhagen in the fall of 1934, August Krogh called 
on me immediately upon my arrival. He wished to apply labelled water 
in his permeability studies. 

I initially intended, upon return to Copenhagen, to do work with 
deuterium on similar lines as later published by Schoenheimer and 
Rittenberg. The possibility of obtaining artificial radioactive isotopes, 
however, induced me to abandon this plan and to concentrate on the 
application of radiophosphorus in biological studies. 



26 ADVENTURES IN RADIOISOTOPE RESEARCH 

Radioactive Phosporus 

As a neutron source we had only radon-beryllium, later radium-beryl- 
lium mixtures, at our disposal. When Niels Bohr celebrated his fiftieth 
birthday, his friends presented him with 600 milligrams of radium, which 
he most kindly put at our disposal. With such modest neutron sources, 
the only tracer of an element of physiological importance which could 
be produced having sufficient activity was radiophosphorus. We irradi- 
ated 10 liters of carbon disulphide from which carrier-free ^^P could be 
easily separated. All our preparations, however, had an activity below 
1 [xc . The first problem attacked was whether the mineral constituents 
of the skeleton are renewed or not during life. Labelled phosphate was 
administered to rats, and the specific activity of their plasma inorganic 
phosphorus and skeleton apatite phosphorus compared. The comparison 
indicated a 30 per cent renewal in the course of the first 24 hours. The 
amount of phosphate involved in this process exceeded twenty times the 
phosphorus content of the blood. Thus a large part of the phosphorus 
present in the soft tissues must have been released and applied in the 
replacement of skeleton phosphorus. This result demonstrated the 
dynamicity of phosphorus metabohsm. These conclusions were pub- 
lished about the same time, in 1935, as the first paper by Schoen- 
heimer and Rittenberg appeared in which they demonstrated the 
dynamic nature of fat depots. It was followed by a great number 
of other most illuminating papers in which deuterium, and later heavy 
nitrogen, was applied as a tracer. Since ^^P has a half-Kfe of fourteen 
days only, happenings through life of a mouse cannot be followed using 
this tracer. However, applying ^^Ca we succeded a few years ago in 
showing that only one-third of the calcium atoms of the skeleton of the 
mouse are replaced during life. 

The above-mentioned first application of an artificial radioactive 
isotope as a tracer was followed by our investigation of whether and to 
what extent the phosphatide molecules of the brain are renewed. These 
investigations were extended to other organs and to the formation 
of labelled phosphatides in the chick embryo following the injection 
of 32p into the fertilized egg. We transfused labelled plasma of a rabbit 
to a sister rabbit and followed the rate of disappearance of the 
labelled phosphatide molecules from the circulation of the second rabbit 
and their accumulation in various organs. The next step was the study 
of the rate of renewal of the ATP, creatine, and similar molecules, partly 
in collaboration with Professor Parnas in Lwow, Poland. With Arm- 
strong and also with Krogh and Hoist, we studied ^sp incorporation in 
dentine and enamel. In one of the early applications (1937), the penetra- 
tion of 32p into yeast cells was traced and shown to be an almost one- 
way process. This investigation was made possible by co-operation with 



A scie>;tific career ^27 

Linderstrom-Lang and Olsen at the Carlsbeig l.aWoiatory. Tlu; 
i'irst investigations of the uptake of ^^P by plants (1936 — 37) was also 
carried out in co-operation with them. 

In 1940 Professor Hasting, who had formerly visited Copenhagen, 
invited me to deliver the Dunham Lecture at Harvard University. 
Denmark was occupied, and messages to the United States could be 
sent only l)y the United States Legation in Copenhagen. When I called 
on the minister asking him to forward a cable to Professor Hasting 
stating, "1 shall be in New York the 21st of June," the Minister remarked, 
"You 'd better write 'I intend to' ". It was a wise remark, as I did 
not succeed in getting to the United States and the Dunham Lecture 
was ultimately delivered by Schoenheimer. 

We observed, w^th Aten, that while phosphate penetrates comparat- 
ively slowly into erythrocytes, it is incorporated very rapidly into labile 
organic acid-soluble molecules. Thus the red corpuscles are a kind of 
trap, though imperfect, for ^^F, a fact which makes it possible to tag red 
corpuscles with ^^p^ re-inject these into the circulation, and from 
the dilution figure calculate the red corpuscle volume of the subject 
in the course of a day. This method of red corpuscle volume determina- 
tion found an extended application. The first clinical determinations 
could be carried out with the minute ^^p activities prepared by us by 
irradiation of carbon disulfide with neutrons emitted by a radium- 
beryllium source. To investigate the formation of phosphatide or of 
casein in the milk of the goat, w^hich was the subject of the dissertation 
of A. H. W. Aten, larger activities were needed. These were prepared by 
Martin Kamen, put at our disposal by the great kindness of Ernest 
Lawrence. He supplied us later also with ^^Na and ^^K. We used these 
isotopes, among other purposes, to study the rate of interchange of 
vascular with extra vascular ions. We were much impressed by the observ- 
ation that within the first minute a very large fraction of the so|lium 
ions of the circulation, for example, was replaced by extravascular 
sodium. Today we know that the exchange-rate values obtained by the 
tracer method supply minimum figures only. 

Max von Laue's and James Frank's Nobel Medals 

My w^ork was interrupted for only one day during the enemy occupa- 
tion of Denmark. When, on the morning of Denmark's occupation, 
I arrived in the laboratory, I found Bohr worrying about Max von 
Laue's Nobel medal, which Laue had sent to Copenhagen for safe-keep- 
ing. In Hitler's empire it was almost a capital offence to send gold out 
of the country, and, I^aue's name being engraved into the medal, the 
discovery of this by the invading forces would have had very serious 
consequences for him. (Three years later the invading army occupied 



28 ADVEXTURES IN HADIOSIOTOPE RESEARCH 

Bohr's institute.) I suggested that we should, bury the medal, but 
Bohr did not like this idea as the medal might be unearthed. I decided 
to dissolve it. While the invading forces marched in the streets of Copen- 
hagen, I was busy dissolving Laue's and also James Frank's medals. 
After the war, the gold was recovered and the Nobel Foundation gene- 
rously presented Laue and Franck with new Nobel medals. 

The Nobel Prize 

In December, 1935, on their journey home from Stockholm, where 
they were presented by King Gustaf V with the Nobel prize, for their 
fundamental discovery of artificial radioactivity, Frederic Joliot-Curie 
and his wife stayed for a while in Copenhagen. It was then that Joliot 
mentioned that he, his wife, and the third French Nobel laureate, Jean 
Perrin, proposed me for the Nobel prize and also that they failed to 
obtain the adherence of the Paris Academy to their proposal — the 
celtium- hafnium controversy was not yet forgotten. During the war 
Niels Bohr with his extreme kindness remarked to one of his friends that 
one of the numerous disturbances created by the war was that I could 
not receive the Nobel prize. The shocking refusal of the acceptance of 
the prize by Domagk, Butenandt, and Kuhn at the order of their ruler 
made the Swedish Academy of Sciences reluctant to distribute further 
prizes during the war. In 1944 the Academy decided, however, to award 
me the prize for 1943. With the war going on, no festivities were held, 
and the prize, contrary to the usual custom, was handed over to me in 
a meeting of the Academy of Sciences by the president. 

Radioactive Tracers in Radiobiology 

In 1940 we got interested, with L. v. Hahn, in the formation rate of 
desoxyribonucleic acid, DNA. While the incorporation of ^^p^ for ex- 
ample, into adenosintriphosphate of the growing liver indicates mainly 
renewal of these molecules and not an additional formation, the incor- 
poration into desoxyribonucleic acid indicates the latter to at least a 
very large extent. 

By investigation of the effect of ionizing radiation on the incorporation 
of 32p into DNA, it should thus be possible to find out if irradiation 
blocks DNA formation. Together with Professor Hans von Euler, we 
studied in Stockholm the incorporation of ^^P into the DNA of the 
Jensen sarcoma of rats and found in the investigated 100 rats 
exposed to Roentgen rays a marked depression of ^^P incorporation, and 
thus a marked depression in the rate of formation of DNA. Similar 
results were obtained when investigating ^^p incorporation into the DNA 
in the various organs of growing rats. Indirect radiation effects 'were 



A SCIENTIFIC CAREER 29 

observed by us as well. These were among the first application of radio- 
active tracers in radiobiological studies. Our joint investigations, among 
others, were extended to the determination of the number of fertilizing 
asp pollen, the atoms of which can be located in a seed. The incorporation 
of 3'-P into DNA of the nucleated erythrocytes of the hen was found, 
in collaboration with Ottesen about the same time, to be quantitatively 
conserved during the lifetime of the erythrocytes, which enabled us to 
measure the life-cycle of the red corpuscles of the hen. 

Prior to and during the war I saw a lot of August Krogh, famous 
physiologist and a man of great kindness, to whom I was much indebted. 
While staying in Stockholm, he wrote down a detailed program of 
further permeability studies in which radioactive tracers would have 
to be applied. It is much to be deplored that he could not witness the 
great further success of his eminent pupil Ussing in this field. 

Radioactive Carbon 

My chief activities since 1943 have been in Stockholm and, for some 
years after the war, in Copenhagen too. During the last years I have 
been attending solely to my laboratory in Stockholm. I extended the 
radiation studies to the measurement of ^'*C incorporation into DNA in 
the organs of growing mice, which was found to be depressed in contrast 
to incorporation into proteins. My colleague Forssberg and I studied 
the effect of irradiation on bicarbonate, glucose, and fatty acid meta- 
bolism and other problems, applying i^C as a tracer. These studies, among 
others, led to the discovery of a fatty acid fraction of the liver having a 
very rapid turnover rate. For the last years we have been interested 
in physiological and clinical problems of iron metabolism. 

In 1953 I had the privilege to deliver the Aschoff Memorial Lecture, 
which is given each year in the University of Freiburg to commemorate 
the great pathologist. Aschoff was not only one of the great pathologists 
of this century but a man of great wisdom and vision. The British patho- 
logist Robert Muir wrote in his obituary- note on Aschoff, published 
during the war, "I think one may say that in the period since Virchow's 
time, he has been the outstanding figure." Aschoff showed some interest in 
our early work with lead and w as quite enthusiastic about the determina- 
tion of the volume of the body water by applying heavy water as an 
indicator, which was the first clinical application of isotopic tracers. 
In my Lecture I mentioned that our investigations had led us to the 
conclusion, not unanimously accepted by the audience, that the forma- 
tion of haemoglobin is not radiosensitive, that so long as erythropoetic 
cells with an incomplete haemoglobin cotitent are present in the bone 
marrow, even if the organism is exposed to Roentgen radiation, additional 
hemoglobin is laid down in these cells. Since then this conclusion has 



30 ADVENTURES IN RADIOISOTOPE RESEARCH 

been fully corroborated by work carried out in our laboratory, and 
especially by the beautiful work carried out in Oxford by Lajtha and his 
associates. 

When we started with Paneth in the first days of 1913 to apply radium 
D as a tracer of lead, the word "isotope" was not yet coined. Groups of 
radioactive substances such as mesothorium and radium, or ionium and 
thorium, w^ere denoted by Soddy as "chemically inseparable elements". 
Much has happened since those days! 



Originally i^ublishcd in Z. (iiwiy. Chem. 82, 322 (J 913) 

1. THE SOLUBILITY OF LEAD SULPHIDE 
AND LEAD CHROMATE 

George Hevesy and Fritz Paneth 
From the Institute of Radium Research of the Vienna Academy of Sciences 

The fourth decay product of radium emanation, RaD, is known to 
exhibit all the chemical reactions of lead ; if RaD is mixed with lead 
or lead salts it cannot be separated from the lead by any chemical or 
physical method^ and if complete mixing of the two substances has 
taken place then the same concentration ratio is maintained whatever 
amount of lead is withdrawn from the solution. Since RaD can be 
determined in much smaller amounts, owing to its radioactivity, than 
lead, it may be employed for the qualitative and quantitative estimation 
of lead to which it has been added ; the RaD is an indicator of the 
lead. 

The lower limit for the qualitative detectability of lead in its most 
sensitive microchemical reaction^ (precipitation of K2PbCu(N02)6) 
amounts to 3 x 10-^ gm ; the limit for quantitative determination lies 
considerably higher and varies with the particular problem ; for ex- 
ample, the solubility of lead carbonate could be obtained from deter- 
minations of the conductance but Kohlrausch^ was able only to make 
an approximate estimate for lead chromate in this way. With the aid 
of RaD as a tracer these solubilities can easily be determined directly ; 
an amount as small as 10"io gm RaD may be measured, by means of an 
ordinary and not particularly sensitive electroscope, if one is content 
to measure the /^-radiation of RaE which comes to equilibrium with 
the RaD after a few weeks. By awaiting the formation of a quantity 
of RaF sufficient for calculating the equilibrium amount, it is possible 
to determine quantitatively even IQ-^^ gm of RaD by means of the a- 
radiation. In radiolead from pitchblende there is about 10"" gm of RaD 
per gm of lead and thus 1 mgm of radiolead can be detected with the aid 
of its ^-radiation ; since much smaller orders of magnitude are involved 



^ A review of relevant experiments is c-ontained in the paper by F. Paneth 
and G. Hevesy in Monatsh. Chem. 42, 1 (1913). 
2 J. Emich, Lehrhuch d. Mikrochemie p. 80 (1911). 
3F. KoHLRAUscH, Z. phys. Chem. 64. 159 (1908). 



32 ADVENTURES IN RADIOISOTOPE RESEARCH 

in the solubilities discussed above we must therefore prepare artificially 
radiolead by the addition of relatively large amounts of radium-D 
to lead nitrate. 



1. DETERMINATION OF THE SOLUBILIIY OF LEAD CHROMATE 

x\bout 0.2 c of emanation was allowed to decay in a closed flask over 
distilled water and the solution thus obtained, containing about 10~^ gm 
RaD in water, was added to a solution of approximately 10 mgm PbClg 
in water. The lead was then quantitatively precipitated with potassium 
chromate, filtered, washed from the filter into a stoppered bottle and 
shaken with about 100 cm^ of distilled water in a thermostat at 25° 
for a period of 24 hr. The mixture was immediately filtered, the first 
portion of the filtrate being rejected because of a possible change 
in its concentration as a result of adsorption on the filter, and 70 cm^ 
of the remaining filtrate was evaporated to dryness on a watch- 
^lass-shaped nickel tray over the water bath. When equilibrium had 
been established between the RaD and RaE the activity on the tray 
was measured. 

The calculation was done as follows : 1 cm^ of the RaD solution used 
for labelling the lead showed (also after establishment of equilibrium) 
a ^-activity of 16.90 arbitrary units and, therefore, the whole solution, 
amounting to 120 cm^, contained 2030 units. This activity had been 
distributed on 9.69 mgm of lead chloride or 11.35 mgm of lead chromate 
and thus one arbitrary unit of RaD was associated with 11.35/2030 = 
= 0.00559 mgm lead chromate. The 70 cm^ of solution which had been 
evaporated had deposited an activity of 0.15 units on the tray and thus 
0.15 X 0.00559 = 0.000839 mgm of lead chromate must be on the tray. 
Hence, the solubility of lead chromate at 25° C is calculated to be 
1000 X 0.000839/70 = 0.012 mgm/1. 

A second experiment with the same solid phase also gave 1 .2 x 10"^ gm/l. 
The first experiments, carried out with smaUer amounts of RaD and 
accordingly with a much lower accuracy, yielded values which varied 
between 3 x 10"^ and 6 X 10-^ gm. Lead chromate is therefore the most 
sparingly soluble lead salt ; only the solubility of lead phosphate is 
of the same order of magnitude. 

Apart from a rough estimate by F. KohlkauschI based on a measure- 
ment of conductance of the saturated lead chromate solution, there are 
no data available on the solubility of lead chr<tmat(^ ; Kohlrausch 
estimates the solubility as 10-* gni/1. 



IF. KOHT.RAUSCH, Z. phys. Chem. 64, 159 (1908). 



THE SOLUBILITY OF LEAD SULPHIDE AND LEAD CHllOMATK 33 

2. DETERMINATION OF THE SOLUBILITY OF LEAD SULPHIDE 

For these experimenls 9.(39 nigin ol' lead chloiide (8.36 mgni in terms 
of sulphide) were labelled with 140 cm^ of another solution of RaD 
which contained 6G.2 arbitrary units per cm^. The lead was then quantit- 
atively precipitated at the boil with a hot solution of NagS, the PbS 
was filtered off, washed and shaken with distilled water as described 
in the case of lead chromate. The filtrate, the first part of which was 
again rejected, was completely clear and colourless ; it contained 
415 arbitrary units of RaD per litre. In this instance one arbitrary unit 
corresponds to 8.36/140 X 66.2 = 9.0 x 10^* mgm of lead sulphide, and 
thus 1 1. of solution at 25° C contained 415 x 9.0 x lO"* = 0.37 mgm 
or 3.7 X 10~^gm. The same value was obtained after filtering the solution 
once again ; other experiments yielded the values of 300 and 320 ar- 
bitrary units per litre, i.e. 2.70 and 2.88 X lO^^g^ j^^d sulphide per litre. 

A part of the lead present in the solution probably occurs as hydroxide, 
owing to hydrolysis, instead of sulphide, as suggested by 0. Weigel^. 
The very weak turbidity obtained when the completely clear saturated 
solution, prepared by shaking water with PbS, is treated with a stream 
of HgS supports this view^ We have therefore determined the solubility 
of PbS in water saturated with HgS ; the solution from w^hich the PbS 
is precipitated cannot be used directly for determining the solubilit\- 
since a portion of the PbS passes as a colloid through the filter ; th(> 
once-filtered PbS, on the contrary, is already freed from the small 
particles passing through the filter and these do not recur when the 
solution is shaken with distilled or HgS-saturated water. In the solution 
which is saturated with H2S and PbS, the concentration of H2S is about 
one thousand times that of the PbS. The solubility of the latter is less 
than the value obtained in distilled water ; calculated on the basis 
of 1 1., the arbitrary activity amounted to 148 and 173 and hence the 
amount dissolved was 1.33 and 1.56 x 10~* gm, respectiv^ely. It is not 
possible to decide with certainty whether there is a decrease in solubility 
due to an increase of the S ion concentration or due to prevention of 
hydrolysis ; the first case, however, is improbable since the decrease 
in solubility is only very slight in proportion to the high concentration 
of HgS. In analytical practice, moreover, this problem need not be 
considered ; it is only of interest to know the amount of PbS which 
is present in solution in a clear filtrate ; our experiments give an average^ 
value for this of 3 X 10"* gm in the absence of HgS and 1.5 x 10^^ 
in a solution saturated with HgS. If the filtrate runs turbid through 
the filter, it is evident that the amount of PbS will be greater. In one 
instance we observed 1 — 2 mgm/1. 

20. Weigel, Z. phy-s. (Item. 55, 293 (1907). 
,3 Hevesy 



34 ADVENTURES IX RADIOISOTOPE RESEARCH 

W. BiLTZ^ determined the solubility of PbS by means of an ultra - 
microscopic method: When two equivalent solutions which produce 
a precipitate are mixed in a series of experiments at increasing dilution 
and the mixture obtained is observed with an ultramicroscope it is noted 
that, beyond the limit of macroscopic differences, the number of suspended 
particles of the precipitate becomes steadily less until, at a certain 
dilution, the mixture appears to be empty or no longer different from 
its components. This limiting value for the disappearance of the un- 
dissolved excess corresponds to the solubility of the substance produced. 
i.e. lead sulphide. Biltz finds a value of 1.3 mgm/1. for the solubilit}- of 
lead sulphide at room temperature. He remarks that the determination 
is made more difficult, in the case of sulphides, because they form 
colloidal solutions which are almost optically transparent at a high 
dilution ; separate particles can of course be produced by adding salting- 
out electrolytes with dissimilar ions but at the same time this may 
cause an increase in solubility. The solubility determined by the ultra- 
microscopic method is therefore probably rather too large. Correspond- 
ingly, O. Weigel^ found that 0.86 mgm of freshly precipitated PbS dissol- 
ved in 1 1. by calculating the solubility of PbS from the conductance on the 
assumption that all the PbS going into solution is hydrolysed. Freshly 
precipitated PbS, however, undergoes a transformation and after about 
20 hr have elapsed the solubility amounts only to about 0.43 mgm/1. 
The PbS used in our experiments was already transformed and the 
solubility of 3 x 10"^ gm in 1 1. which we found agrees very well with 
Weigel's value^. 

RaD is not the only radioelement which can serve as an indicator 
for lead ; careful studies by Fleck^ demonstrate that thorium-B. 
radium-B and actinium-B also cannot be separated from lead. The last 
two cannot indeed be considered for our purposes but thorium-B, with 
its half-life of 10.6 hr, might well be applied with success as an indicator 
for lead. 

Besides lead, we know of two other elements with which a radio - 
element can be used in practice as an indicator, viz. bismuth and thorium. 
The former can be labelled with thorium-C or preferably with RaE,^ 
while the latter may be labelled with uranium-X, radioactinium, radio- 
thorium or, best of all, ionium^. 

1 W. Biltz, Z. phys. Chem. 58, 288 (1907). 

1 O. Weigel, Z. phys. Chem. 55, 293 (1907). 

2 1. BER>fFELD, Z. phys. Chem. 25 (1898) considers the PbS electrode to be a 
reversible electrode of the second kind and calculates the lead ion concentration 
to be 10—* at the PbS electrode at one atmosphere pressure of hydrogen sulphide 
from the electromotive force of the cell Pb | IN Pb(N03)2 | IN NaHS | PbS. 

3 A. Fleck, Proc. Chem. Soc. 29, 7 (1913). 

4 A. Fleck, Proc. Chem. Soc. 29, 7 (1913). 
6 F. SoDDY, Radiochemisiry . London (1911). 



THE SOLUBILITY OF LEAD SULPHIDE AXD LEAD CHROMATE 35 

An advantage of the indicator method is that, irrespective of impuri- 
ties, only the amount of the labelled element is measured, whereas in other 
very highly developed mieroanalytical methods of determination, e.g. 
by employing microweighing, there is always the danger of co-deter- 
mining invisible impurities. Apart from this, the sensitivity of the 
radioactive indicator methods is indeed significantly greater and, assu- 
ming the availability of adequate amounts of the radioactive substance, 
can ])e increased almost without limit. 

Summary 

The solubility- of lead chroniate at 25° in pure water has been determined as 
1.2 X 10~5 gm/1-; for lend sulphide at 25° in puie water and in HjS-saturated water 
the values were 3 x 10~* and 1.5 X 10* gm/l. respectively; RaD was used as 
a tracer for lead. 



Originalh published in Z. phys. Chem. A 171, 41 (1934) 



2. PLATINUM BLACK 

G. Hevesy and T. Somya 
From the Institute of Physical Chemistry, University of Freibuig 

For the preparation of platinum black, which is used in hydrogen 
electrodes and for other purposes, the electrol3^sis of hydrochloric acid 
solutions of platinum containing lead acetate is employed. The question 
then arose as to whether the presence of lead in the solution is essential 
to the preparation of good platinum black and. if so, as to the part 
played by the lead. To obtain an answer to the first question, we have 
electrolysed both hydrochloric acid solutions containing only platinum 
chloride and others containing also small quantities of lead. It was 
shown that platinum black cannot be obtained successfully b}^ the 
electrolysis of a solution containing only platinum. On the contrary, 
a grey or light brown deposit is always obtained. On the other hand, 
the preparation of platinum black is accomplished from solutions which 
contain a corresponding amount of other heavy metals in place of 
lead. After this observation we proceeded to study whether lead is 
carried into the deposit when a solution containing lead is electrolysed 
and, if so, in what amount and form. 



DETERMINATION OF THE LEAD CONTENT OF PLATINUM BLACK 

Since the detection of small quantities of lead in platinum is very 
tedious we have made use of a radioactive tracer method. A known 
amount of lead acetate labelled with thorium-B was added to the 
platinum chloride solution and the lead content of the platinum black 
deposited on platinum electrodes having a surface area of 15.07 cm^. 
at a current density of 10 mA/cm'-. was determined by measuring the 
intensity of the a-radiation emitted by the deposit. The amount of lead 
was calculated from this intensity measurement as follows : From the 
same radioactive lead acetate solution, of which a known volume w^as 
added to the platinum chloride solution, lead peroxide was precipitated 
(see below) after adding nitric acid and a further amount of inactive 
lead acetate and the a-radiation of this precipitate was compared with 



IM-ATIXTM BLACK 37 

that of the platinum black. Now if eare is taken that the thickness of 
the deposits attains the range of the a-radiation in the material, which 
is 12.8 fi in lead and 30.6 jj, in lead peroxide, then the activity will 
provide a simple measure of the lead content. Denoting the activity of 
the platinum black electrode by 8.^, that of the lead peroxide electrode 
of the same size by 8^^ the density of platinum (21.3) by d^, the density 
of lead peroxide (8.9) by (h, the range of a-radiation in platinum (12.8 [.i) 
by i?i, the range of a-radiation in lead peroxide (30.6 [a) by R^, the number 
of grammes of lead in 1 cm^ of the active lead acetate solution by p and 
the number of grammes of lead in the 2.5% lead acetate solution by P. 
then the required lead content of the platinum black, x, expressed as 
a percentage, is given by 

X = 8^pR^d^{ii\. wt. of Pb)lOO/iS'2/;Pi^jr/i(mol. wt. of PbO.,) 

Therefore p was chosen to differ from P because it had been found 
preferable to produce the lead peroxide deposit from a solution with 
a lead content higher than that for the platinum black coating. The lead 
content of the platinum black, as determined, is found in the Figures in 
Table 1. It may be seen that the lead content of the platinum black rises 
sharply with increasing lead acetate content of the platinum chloride 
solution. 

Tablk 1. — Lkad Content of Platintjm Black as a FrNCTioN 

OF THE Lead Acetate Content of the Electboly.sed 0.2 N HCl 

Solution Containing 3°o PtCl^ 



Lead Acetate Content 


Le:id Content of the 


of the Solution 


Platinum Black 


(%) 


(%) 


1.22 


0.035 


1.34 


0.815 


1.44 


1.5 


1.9 


7.1 



In order to decide whether the lead found in platinum black is present 
in solid solution or not we have compared the line distances obtained 
on Debye-Scherrer diagrams for different platinum black samples with 
those of pure platinum. The difference, as is evident from Table 2, was 
shown to be vanishingly small and therefore it must hv assumed that 
the large majority of the lead occurring in platinum black is not present 
in the form of a solid solution. The measured line distances underwent 
a considerable increase when the sample was heated. Thus, the second 
sample recorded in Table 2 showed, after heating for 16 hr in a vacuum 
to 500°C, a line distance of 124.7 mm ; aflcr 44 hr heating at 025'^ 
the distance was 125.2 mm. 



38 



ADVENTURES IN RADIOISOTOPE RESEARCH 



Table 2. — Line Distance of the (422) Interference of Platinum 

Black 



Lead content of the 

platinum 

(%) 


Line distance before 

heating 

(mm) 


Line distance after 

heating in vacuum 

(mm) 




1.5 

7.1 


124.3 
124.4 
124.1 


124.6 (500°) 
125.2 (625°) 

125.7 (625°) 



Heating to still higher temperatures resulted in a considerable evapor- 
ation of lead, as shown in Table 3. With regard to the values in Table 2, 
it should be mentioned that it was not possible to assess the line distance 
in the case of platinum grey (lead-free platinum deposit) with sufficient 
accuracy. The number 124.3 in the second column thus refers to platinum 
wire whereas the corresponding value in the third column was indeed 
obtained from platinum grey. After heating, the platinum grey did of 
course yield lines of adequate sharpness. The exposures for the Debye- 
Scherrer diagrams were obtained with the aid of the precision camera 
described by Sachs and Weerts^ ; platinum wires 4 mm thick and 
coated with platinum black were used for the exposures. A Metalix 
tube with a copper a/?ti-cathode was used as the source of radiation and 
was operated for as long as 13 hr at45kV and 20 mA. The lead content 
present as a solid solution was calculated by means of Vegard's law, 
according to which the lattice constant of the solid solution is 

a = (3.905^1 -f 4.93ro)/100 

where a is the length of the side of the unit cell of the alloy, q is the 
number of atoms per cent of platinum and Co, for lead. The calculation 
showed that, of a total of 1.5 per cent lead, only 0.2 per cent was present 
in solid solution after heating (Table 2) and of the 7.1 per cent lead in 
another sample only 0.3 per cent was similarly in solid solution. 

It is hoped to study in more detail, by means of radiographs of platinum 
black containing thorium-B, the distribution of lead in platinum. 



THE EFFECT OF HEATING ON THE LEAD CONTENT OF PLATINUM 

BLACK 

It has already l)een mentioned that considerable amounts of lead 
(Ma])orated when platinum black was heated at higher temperatures. 
in a more detailed study of this point the alphaactivity of platinum black. 



iSack^ and \Vep:rts. Z. I'hy-s. 60, 481 (1930). 



rLATIXlM BLACK 



39 



obtained l)y llie electrolysis of solutions containing lead acetate, labelled 
with thorium B, was determined before anrl af1(>r heating. The results 
of this experiment are seen in Table 3. 



Table 3. — Effect of Heating for 16 hr in a Vacuum on the Lkad- 

CONTENT OF PlATINUM BlACK ORIGINALLY CONTAINING 1.5% LeAD 



Tempenuuix- ( C) 6UU— (310 6^0-600 j 710— 72 j 


Loss of lead calculated from the 
decrease in a-activity (%) 


2 


53 


85 



The loss of lead is not entirely due to evaporation but partly also to 
diffusion of the lead contained in the platinum black into the platinum 
foil on which the coating was deposited. 

Differentiation between the loss by evaporation and diffusion, is 
possible by making use of the y-radiation instead of the a-radiation 
for making the comparison ; whereas the amount of lead removed by 
diffusion weakens the a-radiation to the same extent as does the lead 
disappearing through evaporation, this is is not the case when the 
y-activity is measured. For example, the decrease in y-radiation after 
16 hr heating at 685 to 700°(" amounted to only 42 per cent and thus 
considerably less than the decrease in a-radiation. 



THE QUALITY OF THE VARIOUS SAMPLES OF PLATINUM BLACK 



An attempt was next made to measure the easily traced adsorption 
of thorium B and thorium C from solutions of these radio-elements with 
a vieW' to assessing the quality of the platinum black. Yet great difficulty 
was encountered in obtaining reproducible results. This method was 
therefore relinquished. It was then thought that a simple measure of the 
quality of the various platinum black coatings could be obtained by 
preparing hydrogen electrodes from the various platinum black samples 
and comparing their potentials. It was shown, however, that the potential 
of all the hydrogen electrodes prepared in this way m' as always the same 
within the experimental error of about 1 mV. We then changed over to 
determining how strongly the various samples of platinum black could 
be polarized with the same cathodic loading and to making use of the 
difference in polarizability as a measure of the quality of platinum black. 
The polarization was performed in N sulphuric acid solution with a 
current density of 20 mA/cm^ at room temperature for a period of 45 
min ; the area of one side of the electrode amounted to 1 cm^. The polariz- 



40 ADVENTURES IN RADIOISOTOPE RESEARCH 

ation potential was measured by using a normal hydrogen electrode. 
A constant A^alue of the polarization potential was established after 
about 30 min. The result of the measurements is evident in Table 4. 



Tablk 4. — Cathodic Polarizability of Platintm Deposits Obtained 
FROM Solutions with Different Lead Contents. Polarization Current 

Density 20 mA/cm^ 



Lead content of the 
electrode (%) 

Polarization potential 
(mV) 



7.1 1.5 0.1.3 0.035 

84.4 77.5 81.0 97.9 103.7 



The least polarizable and, therefore, ihe one of highest quality is 
platinum black with a lead content of 1.5 per cent, and it is interesting 
to notice that this sample of platinum black is identical w-ith that 
obtained by electrolysing a solution containing 1 part of platinum chloride 
and 0.008 parts of lead acetate in 30 parts of water, and that Lummer 
and KuRLBAUM a long time ago used the electrolysis of a solution of 
Ihis composition for preparing platinum black. This set of directions is 
also included in the Textbook of Practical Physics by Kohlrausch and 
other similar works. 

Attempts were then made to heat the electrodes before they were 
polarized. In all instances the heating spoiled the quality of the platinum 
black. After heating the electrode containing 1.5 per cent lead for 16 hr 
at about 610°C the polarization potential rose from 77.5 to 88.3 mV, 
and after 16 hr heating at about 700° it became 183 mV. 

( -hanging the current density from 10 to 30mA/cm2 when preparing 
the platinum black had no detectable effect on the quality. 



CONNEXION BETWEEN THE PARTICLE SIZE AND QUALITY OF 

PLATINUM BLACK 

The particle size of the platinum black was determined from the half 
breadth of the X-ray lines in accordance with Brill's method^. The 
Debye-Scherrer camera used for this purpose had a diameter of 5.73 cm. 
The diameter of the platinum wire covered with platinum black was 
0.34 mm. Lines of the (220) and (311) faces were used for the investigation. 
The results of this study are shown by the data in Table 5. 



IK. JiRiLL, KuUuid Z. 55, 104 (1931); Z. Krist. 74, (147 (1930). 



PL.A.TINUM BL.A.CK 



41 



Table 5. — Dependence of the Particle Size of Platinu.m Black on 

ITS Lead Context 



Lead content of 
platinum (%) 


Particle 


size (A) 


Calculated from 
the (220) line 


Calculated from 
the (311) line 


7.1 


62 


64 


1.5 


81 


75 


0.15 


68 


68 


0.035 


58 




; 

1 


61 


57 



It is evident from this table that the platinum black sample whicli 
has been found to have the best quality is distinguished by having the 
largest particle size. 



PREPARATION OF PLATINUM BLACK FROM SOLUTIONS CONTAINING 

GOLD 

It is evident from Table 6 that platinum black of good quality was 
prepared also from platinum chloride solutions which contained gold 
instead of lead and was deposited at a current of 30 mA/cm^. 



Table 6. — Cathodic Polarizability of Platinum Deposits Prepared 

FROM Solutions Having Various Gold Contents. Polarization 

C URRENT Density 20 rnA/cm^. Electrolyte 1 N HoSO^ 



Platinum content of the 
solution from which the 
platinum black was ob- 
tained (%) 


Gold content of 

the solution 

(%) 


Polarization 

potential 

(mV) 


0.14 
1.8 
1.8 
1.8 


0.9 
0.1 

0.01 
0.0001 


76.9 

78.7 
77.7 
86.4 



We have also prepared platinum black from platinum chloride solutions 
which contained thallium, cadmium or zinc in place of lead. Whereas 
thallium can substantially replace lead as far as the appearance of the 
deposit is concerned, the behaviour with cadmium is different inasmuch 
as a solution which contained about 0.02 per cent cadmium chloride 
yielded a fine black deposit while the electrolysis of solutions which 
contained only about 0.01 per cent cadmium chloride yielded a grey 
deposit instead of platinum black. Electrolysis of platinum chloride 
solutions containing zinc yielded a grey deposit in all cases. We then 



42 ADVENTURES IX RADIOISOTOPE RESEARCH 

fit tempted to electrolyse hydrochloric acid solutions of pure platinum 
chloride at a high current density, i. e. at 100 mA/cm^ and above. With 
this heavy loading it was no longer possible to obtain an adherent deposit. 
The deposited platinum powder fell into the solution, harl a black-grey 
appearance and was extraordinarily fine grained. 



Summary 

The platinum black obtained from platinum chloride solutions containing lead 
in accordance with the directions of Lummer and Kurlbaum, contains con- 
siderable amounts (1.5 per cent) of lead. Variation of the lead content of the 
platinum black with the lead content of the solution subjected to electrolysis 
was observed. 

The electrolysis of a solution containing 1.9 per cent of lead acetate yields 
a platinum black which contains 7 per cent of lead. By assuming the validity 
of Vegard's additive law for the lattice dimensions it is found that the greater 
part of the lead present in platinum black does not occur in solid solution. 

Of the various samples of platinum black the one prepared in accordance 
with the directions of Lummer and Kuklbaum was the least electrolytically 
polarizable and thus the most perfect. A determination of the paiticle size of 
the platinum black samples by means of the half-width of the X-ray interferences 
resulted in the fact that the best platinum black sample had the largest particle 
size. 

Platinum, black was also prepared from platinum chloride solutions which 
contained other added metals instead of lead. 



Originally communiratod in Ndtnre, 128, 1038 (1931). 



3. LEAD CONTENT OF ROCKS 

G. Hevesv and R. Hobbie 
From the Institute of Physical Chemistry of the University of Freiburg 

In recent years various geochemical problems have arisen which make 
it important that our scanty knowledge of the lead content of rocks 
should be amplified and made more precise. To this end we have deter- 
mined the lead in a series of samples, representing in all about 220 rocks, 
some of which we owe to the kindness of Prof. Arthur Holmes of 
the University of Durham. 

The sample to be analysed was brought into solution ; silver sulphate 
was added and the silver and lead present in the solution were simultane- 
ously precipitated as sulphide. The precipitate was them brought into 
solution and the lead deposited electrolytically as peroxide. That the 
deposit was actually lead peroxide was confirmed by a colorimetric 
test, tetramethyl-diamino-diphenylmethane being added to the solution 
of the deposit. To ascertain that the total lead content was actually 
recovered, we used the method of radioactive indicators. We added to 
the rock sample a known amount of the lead isotope radium D, prepared 
from radium emanation, and checked the yield obtained by measuring 
the activity of the lead peroxide deposit. As the purest chemicals com- 
mercially obtainable were found to contain quantities of lead that 
would have influenced our results, all the chemicals used were first 
purified from lead. Moreover, every precaution was taken to avoid 
contamination of the samples by dust which might have contained 
traces of lead. The results obtained are listed in Table 1. 

The average value found is 16xl0~^ gm lead per gm rock, a some- 
what larger value than that given by Clarke and Steiger^ who found 
7.5xl0~6 gm per gm rock. As shown in the communication lliat 
follows, the amount of lead accumulated in the rocks since the solidifica- 
tion of the earth's crust (as a result of the decay of uranium and thorium) 
is very much smaller. Thus, as between the atomic weights of rock- 
lead and ore-lead we have in most cases to expect differences only in the 



^Clarke and Steiger, ./. Wash. Acad. Set. 4, -IS (1014). 



44 



ADVENTURES IN RADIOISOTOPE RESEARCH 



Table I. — Lead Coxtent of Igneoxts Rocks 



Rock Types 



gm Lead per 
gm Rock 



Basalt, Giant's Causeway 4 X 10"" ^ 

Gabbros and related types (composite of 67 samples) 5 X 10"^ 

Essexites and related types (composite of 40 samples) 10 X 10~^ 

Shonkinites (average of 2 samples) 10 X 10~^ 

Soda-granites and soda-syenites (composite of 2(5 

samples) U X 10"" ^ 

Potash-granites and potash-syenites (composite of 24 

samples) 14 X 10~ ® 

Amphibolite, inclusion in Kimberlite, Wesselton 

Mine, S. Africa 15 X 10^ « 

Kimberlite ('basaltic ' type). Dyke from 1350-foot 

level, Dutoitspan Mine 16 X 10~ " 

Lherzolite, Baltimore, Maryland 19 X 10~" 

Granitic rocks (composite of 58 samples of widely 

different localities) ' 30 X 10 « 

Dunite, Jackson Co., Nortli Carolina 42 X 10"'' 



second decimal place. That the ore-lead must have been formed in 
the ancestral sun, or during the events that attended the birth of 
the solar system, was already pointed out some years ago b}^ Prof. 
Holmes^ . 

We conclude from the above determinations that the greater part of 
rock-lead is also of the same origin. Although acid rocks, which have a 
relatively high uranium and thorium content, are found to contain more 
lead than basic rocks, this difference is not to be interpreted as an 
argument in favour of the radioactive origin of the whole of the lead 
in rocks, but an expression of the fact that lead, like uranium and thorium, 
shows a marked affinity to siliceous magmas. 

It is of interest to compare the lead content of basic and ultrabasic 
rocks with that of meteorites as determined by Noddack'^. Tlie lead 
content of stony meteorites is near that of basalt and average gabbro, 
and is markedly lower than that of terrestrial ultrabasic rocks. The lead 
content of iron meteorites, as confirmed in this laboratory, is about ten 
times greater than that of stony meteorites, while that of troilite (the 
high lead content of which was predicted by Prof. V. M. Goldschmidt) 
is more than a hundred times greater (700xlO~^). These results show 
1 hat when the earth was formed the silicate shell received only a modest 



^Holmes, Nature 117, 482 (1926). 
2NODDACK, Die Naturwiss. 18, 761 (1930). 



LEAD CONTEXT OF ROCKS 



45 



Takli: it. — Li:ad Content of Basic and Ultrabasic Rocks and 

OF Mf:tkorttks 





gm per gtu 
Rock 


Gabbros (average) 

Kimberlite 


5 X 10- « 
16 X 10- « 
19 X 10-« 

5 X 10" « 


Lherzolite 

Stony meteorites (average) 


Iron meteorites (average) 


50 X 10- « 





sharp of" the total lead available lor partition, and that this uneven 
distribution has so far been compensated only slightly by the formation 
of lead from radioactive decay. 



46 ADVENTUKES IN RADIOISOTOPE RESEARCH 



Comment on papers 1,2,3 

Radioactive tracers aio very often applied in the solution of problems which 
can only he solved by making use of this device. Often this method is, however, 
applied not as a necessity but for the sake of convenience, for facilitating the 
solution of problems which can be solved by other methods as well, though moic 
tediously. An example of the latter is the determination of the lead content of 
platinum black described in paper 2. By making use of labelled lead the analysis 
and the behavioui' of platinum black under the effect of heat, of electrolytic 
polarization and so on could be carried out without even dissolving the sample 

The determination of the solubility of lead sulphide and chromate in water 
discussed in papers is a border case. The writer of the well-known text-book on 
physical measurements, Kohlrausch, succeeded in calculating the solubility of 
lead carbonate from the electrolytic conductivity data, but he arrived in the 
case of lead chromate at a rough, estimate of its solubility only. 

In the determination of the lead content of rocks by making use of isotope 
dilution control an isotope of lead is necessarily to be used. This investigation 
was the first application of the isotope dilution method. Being interested in the 
abundance of the elements, we determined various constituents of an artificial 
air crust sample prepared by my colleague, the well-known mineralogist Schneidei- 
hohn, who disposed over a very extended collection of rock and mineral samples. 
We usually applied the method of analysis of X-ray spectroscopy using a secondary 
X-radiation method worked out in the Freiburg laboratory in which these in- 
vestigations were carried out. 

Knowing the intensity ratio of two very closely situated X-ray lines, for example 
that of HfLj andLu/?2' ^Y adding to a pulverized sample to be analysed a known 
amount of LuOg we can, after taking an X-ray spectrum, calculate from the 
intensity ratio of the 2 above-mentioned lines the unlinown hafnium content of 
the sample. When the X-ray spectrum is excited by cathode rays the sample 
gets hot and sputters easily ; for this reason a continuous X-ray spectrum is 
first produced on a metallic tungsten surface and the sample irradiated by this 
continuous rontgen radiation. Heating of the sample and consequent sputtering 
is now avoided. The application of the method in the above-mentioned case 
assumes the absence of significant amounts of the rare lutetium in the sample 
investigated. 

The sensitivity of this method (in the twenties of this century, when we apphed 
it) did not suffice to determine the very small amounts of lead present in rock 
samples and correspondingly we embarked on a chemical determination of lead 
controlled by making use of isotope dilution. This device, discussed further on 
p. 9G, proved to be a very useful one in chemical analysis. 



Originally published in Kgl. Dan-ske VidenshaherneN Selskab. Maihcmatisk-fysiske 

Meddeleher. 14, 5 (193G) 

4. THE ACTION OF NEUTRONS ON THE RARE 

EARTH ELEMENTS 

G. Hevesy and Hilde Levi 
P'loni the Institute of Theoretical Physics, University of Copenhagen 

The action of neutrons on the rare earth elements can be followed uj) 
in two ways : by investigating the radioactivity induced in these elements 
under neutron bombardment, and by observing their absorbing power 
for a beam of slow neutrons. In this paper both these lines of attack 
will be discussed for the rare earth group and for yttrium and scandium. 



ARTIFICIAL RADIOACTIVITY OF THE RARE EARTH ELEMENTS 

The artificial radioactivity of some of the rare earth elements was 
investigated by Amaldi, D'Agostino, Fermi, Pontecorvo, Rasetti 
and Segre (1), others were investigated by ourselves (2) by Sugden (3) 
by Marsh and Sugden (4) by McLennan and Rann (5) and by E. 
RoNA (6). The neutrons used were produced by the action on beryllium 
of the a -rays from radium emanation and were in many cases slowed 
down by inserting layers of paraffin 10 — 20 cm thick in the path of 
neutrons ; a Geiger— MItller counter was used to measure the activi- 
ties obtained. 

Scandium 

A sample of scandium oxide prepared by Prof. Sterba-Bohm and 
kindly presented to us by Prof. Honigschmid, who used the preparation 
in determining the atomic weight of scandium, was activated for a few 
days using an emanation-beryllium source of 200 — 300 MC. The oxid(> 
was then dissolved in dilute hydrochloric acid and 100 — 150 mgm sodium 
chloride as a carrier of ■^^K (cf. p. 48) and the same amount of calcium 
oxide were added. The filtrate obtained after precipitation with carbon- 
ate-free ammonia was treated with oxalic acid and the calcium oxalate- 
formed was removed. The sodium chloride which had been added was 
recovered, after the removal of the ammonium chloride content of 1 he 
last filtrate, by evaporation and ignition. The activities of the three 



48 ADVENTURES IN RADIOISOTOPE RESEARCH 

fractions, those of scandium oxide, sodium chloride, and calcium 
oxalate, were then determined. The two first mentioned preparations 
were found to be active, the activity of the scandium oxide decaying 
very slowly and that of the sodium chloride fraction having a half-life 
of 10 to 16 hours. The activities are due to the formation of |fiSc and 
t|K respectively ; the reactions leading to these products are 

liSc + Jn = IfSc 
and 



45 
21' 



Sc + on = ilK + la 



The mass numbers occuring in these equations follow from the fact 
that scandium has only one stable isotope, ^^Sc. The calcium oxalate 
investigated was inactive ; we are thus unable to find any evidence 
for the reaction IfSc + Jn = loCa -{-\B. which possibly takes place also. 
The activity which cannot be separated from scandium is presumably 
due to iiSc ; most of this activity decays with a period of about two 

months 

While \IK emits hard ^-rays having a half value thickness of 0.19 
gm/cm2 Al, 2iSc emits soft /^rays with a half value thickness of 0.01 gm/ 
cm2 Al. 

Yttrium 

We investigated (2) samples of yttrium oxide kindly given us by the 
late Baron Auer v. Welsbach, by Prof. Prandtl, and by Prof. Rolla. 
The two first named preparations were used some time ago by Honig- 
SCHMiD to determine the atomic weight of yttrium and investigated 
by one of us on that occasion by X-ray spectroscopy. While the investi- 
gation of Baron Auer's preparation revealed the presence of some 
dysprosium, that of Prandtl was found to be of the highest purity. 
The great purity of this preparation and of that of Rolla was also 
shown by their behaviour under neutron bombardment : No initial 
decay with the period of dysprosium (2.5 h.) could be observed, the sole 
period being one of 70 h., which we found to be the period of decay of 
yttrium. Auer's preparation decayed initially with a half-life of 2.5 h.. 
which was obviously that of dysprosium ; but afterwards it showed a 
70 h. period like the other preparations. The molecular volumes of 
corresponding compounds of yttrium and dysprosium are only very 
slightly different*, so these elements are unusually closely related chemi- 

* The volumes of the octahydrosulfates differ by less than 0.8% (G. v. Hevesy, 
Z. anorg. Ch. 147, 217 ; 150, 68 (1925) and the ionic radii by about the same amount 
(V. M. GoLDSCHMiDT, Ullrich and Barth, Oslo. Acad. Proc. Nr. 5 (1925). 



THE ACTION OF NEUTRONS ON THE HARE EARTH ELEMENTS 



49 




10 days 



Fig. 1 



10 days 



^ a) Decay Curve of a Pure and an Impure Yttrium Preparation, 
b) Samarium Decay Curves (the two days' period only; the weak period 

of 40 min is not visible;. 



cally and their separation is attended with very great difficulties. Figure 
1 shows the decay of a pure preparation and of one containing some 
dysprosium . 

Since yttrium has only one stable isotope, ^^Y, the artificial radio- 
activity obtained from it is presumably due to the formation |^Y. We 
find the intensity of the yttrium activity to be 0.005 times as large as that 
of dysprosium, both preparations having been activated until saturation 
was obtained in a paraffin block of 30x30x25 cm edge ; the neutron 
source was placed on the top of the preparation which w^as covered by 
a thin shield of paper. The ^-rays emitted by yttrium are absorbed 
1() half of their initial value bv 0.6 mm Al. 

Lanthanum 

Marsh and Sugden (4) find 1.9 days as the half-life of lanthanum 
and for the intensity of the /?-rays emitted, a value amounting to 35% 
of that observed for the activity of praseodymium. As we find a value of 
22 for the ratio of the radiation intensities of dysprosium and praseody- 
mium, the lanthanum activity works oul at 2.0% of that of dysprosium. 



4 Tlcvcsy 



50 ADVEXTTEES IX RAmOISOTOPE RESEARCH 

As lanthanum has but one stable isotope, ^^^ha, the activity obtained 
is presumably due to the formation of ^|!^La. Fermi's coefficient a, 
indicating the increase in activity when the bombarding neutrons 
are slowed down by a thick layer of paraffin or other hydrogen - 
containing substances, instead of being allowed to impinge directly from 
the beryllium source on to the substance to be activated, was found 1o 
be 12. 

Cerium 

No activity was observed after Ijomljardment of cerium for several 
days with a neutron source of few hundred millicurie. 

Praseodymium 

Amaldi, Fermi, and others (1) found the artificial radioactivity of 
praseodymium to decay with a 19 h. period, the same value being found 
later by other experimenters (4), (5). Although only one stable isotope 
of praseodymium is known. ^^^Pr, the above-mentioned investigators 
found a second period of decay (5 min) which in contrast to the first 
period is not hydrogen-sensitive. * 

Neodymium 

Fermi and his collaborators (1) found that activated neodymium 
decays with a period of 1 h. ; we find (2) that this activity is 2500 times 
as small as that of dysprosium. Marsh and Sugden (4) found no activity, 
while according to McLennan and Rann (5) the half-life is 35 min. 
Neodymium has the stable isotopes 142, 143, 144, 145, 146 and 148, and 
the activity observed is presumably due to the formation and decay of 

497 
fiO-l 



"Wd 



Samarium 

The artificial radioactivity of samarium decays, as was found ))y 
Fermi (1), and later by us, with a period of 40 min. We find (2) the 
intensity of the activity to be 0.6% of that of dysprosium. Samarium has 
furthermore, as was first noticed by Marsh and Sugden (4), a much 
longer period as well. We determined the period of this isotope to be 
2 d., as can be seen from Fig. 1 and found its intensity to be ~ of that 
of dysprosium, i.e. 2.0 on our relative scale. Samarium has the stable iso- 
topes 144, 147, 148, 149, 150, 152, and 154, and it is not possible to deter- 
mine the mass number of the active samarium isotopes with certainty. 



THE ACTIOX OF XEITROXS ON THE RARE EARTH ELEMENTS 51 

A very intense aclivity was ol)lained by Sugden (3) on bombarding 
europium with slow neutrons. It decayed with a period of 9.2 h. The 
intensity of the europium radiation was found by us (2) to be 80% 
of the dysprosium radiation emitted l)y the same amount of dysprosium, 
both preparations being activated until saturation was reached. Care 
was taken, too, that the neutron beam was weakened only to a small 
extent by the activation process, i.e. very thin layers were activated. 
Europium has two stable isotopes 151 and 153 and the activity is 
possibly due to the formation of ^^^Eu. Th.e europium : dysprosmm 
activity ratio is found to be smaller for thick layers than for thin 

The value 40 was found for the hydrogen-effect, a. The half-value 
thickness (2) of the ^-rays emitted is 0.02 cm Al, and it was concluded 
from absorption measurements that energies up to 2.0-106 eV occur.* 
In addition, y-rays have been detected which are little absorbed by 4 
mm. lead. 

Gadolinium 

Fermi and others (1) found gadolinium to decay with a period of 
8 h. after neutron bombardment. McLennan and Rann (5) found a 
half-life of 6.4 h. and twice the intensity found for neodymium. The 
combination of the last mentioned figure with our intensity data leads 
to an intensity value which is 250 times as small as that observed for 
dysprosium. Marsh and Sugden (4) could not find any activity. 



Terbium 

The activity of terbium (3) decays with a period of 3.9 h. As this ele- 
ment has only one stable isotope, ^^Tb, the activity observed is presum- 
ably due to the formation and decay of TsTb. The intensity of the 
radiation (2) observed is 2.5 per cent of that of dysprosium. 



Dysprosium 

The activity of dysprosium (2), (4) decays with a period of 2.5 h. 
and is the strongest yet observed in the domain of artificial radioactivity. 
We have therefore chosen it (2) as a standard of comparison for the 

* R. Naidxj and R. E. Siduy {Proc. Roy. Soc A 48, 332, 3(5) by using a cloud 
chamber determined recently the energies of the ^-ray spectra and found that 
the maximum energy lies at 1 .3 ■ 10« eV, while the upper limit of the spectrum 
is 2.6 • 106 eV. 



4* 



52 ADVENTURES IN RADIOISOTOPE RESEARCH 

activities of the rare earth elements : we denote the intensity of dys- 
prosium arbitrarily by 100. It is of interest to remark that the 2.3 min 
activity of silver, which is considered a very strong activity, is 12 times 
as weak as the activity of an equal amount of dysprosium. The hydrogen 
effect (a) was found to be 100, the half-value thickness of the /?-rays 
emitted was 0.025 cm Al ; and the upper limit of the continuous /5- 
spectrum concluded from absorption measurements with aluminium 
has an energy of 1.4 • 10^ eV (2).* Dysprosium is one of the commoner 
rare earth elements of the yttria group and as it is very strongly active, 
activated samples of rare earth elements denoted as "erbia", "holmia", 
"yttria", etc. often decay with the period of dysprosium. 



Hoi 



nuum 



We found (2) the activity of holmium to decay with a period of 35 h., 
while E. RoNA (6) recently found the value of 33 h. The half-life of 2.6 h. 
measured by Marsh and Sugden (4) and later by McLennan and Rann 
(5) is presumably due to the presence of dysprosium in their preparations ; 
some of our impure preparations, too, showed an initial decay with the 
period of dysprosium. The samples of holmia investigated were given 
us by the late Baron Auer. Holmium has one stable isotope, 165 ; the 
activity observed is therefore presumably due to the decay of ^evHo, 
the intensity of the activity observed being 20 per cent of that ol 
dysprosium. The hydrogen-effect (a) is much smaller (2) than that of 
dysprosium; the half value thickness is 0.04 kgm/cm^Al ; and the upper 
limit of the /^-ray spectrum has an energy of 1.6 • 10^ eV. 

Erbium 

Erbium has a very weak activity of similar intensity to the 40 min 
samarium radiation, decaying with a 7 min period according to Marsh 
and Sugden (4), and with a 4.5 min according to McLennan and Rann 
(5). A second period (2) was found by us to be 12 h. ; the period of 2.5 h. 
ascertained by Sugden (3) using a commercial preparation is presum- 
ably due to the presence of dysprosium, and that found by Marsh and 
Sugden (4), 1.6 d., to the presence of holmium in the sample investi- 
gated. Recently Rona (6) has given the value of 13 h. for the longer 
period. The intensity (2) of the longer period of erbium is 0.35 per cent 
of that of dysprosium, and the half- value thickness of the ^-rays emitted 
is 0.03 cm Al. 



* R. Naidu and R. E. Siday, loc. cit. found that the maximum energy lies 
at 0.75 • 108 (.y. while l.S • ]0« eV is ihe upper limit. 



THE ACTION <)1" XKITROXS OX THE RAHE EAKTH ELEMEXTS 



53 



This element shows an activity having a long- life as first stated by 
RoNA (6) who finds 1hat the /?-rays emitted are half absorbed by 0.015 
cm Al. E. Neuningek and 1^]. Roxa* found recently a period of 4 



300 

100 

50 

30 



lOOr 



50 



Lu 



\ 



30 



20 



10 



V- 



Lu 



\' 



10 



20 dav5 



10 20 30 «) 50 



100 davs 



Fif,'. 2. I.utecium Decay Curve. 

a) showing the sliort period and the beginning of the 7 days' period. 

b) showing the measured points for the 7 days' period and the curve 

obtained after subtracting the residual activity. 



(± ^2) months. After bombarding 100 mgm TmOg, kindly lent us by 
Prof. Jantsch, w ith about 100 mc for 23 days we obtained 60 counts per 
minute, while 100 mgm of dysprosium activated to saturation with the 
same source gave about 4000 counts per minute. The activity of this 
preparation decayed with a period of about 3.5 months ; a thulium 
preparation activated to saturation would therefore exhibit an activity 
about i/j„ of that of dysprosium. 



Ytterbium 

The activity of ytterbium (2), (3) decays with a period of 3.5 h. 
As ytterbium has the isotopes 171, 172, 173, 174, and 176, it cannot be 
decided whether the activity is due to the formation and decay of ^7oYl> 



*E. Neuninger and E. Ro>a, Wien. Anz. 73, J",;) (1936). 



54 ADYEXTIEES IX KADIOISOTOPE KESEAECH 

orof ^^^Yl). The ytterbium radiation (2) is somewhat weaker than that 
of erbium, and amounts to 0.3 per cent of that of dysprosium. The 
half-value thickness of the ;5-rays emitted is 0.04 cm Al. 

Lutecium 

Lutecium (cassiopeium) exhibits an activity of fairly long life (2), 
namely one decaying with a period of 6 — 7 d.. and having an intensity 
of L4 per cent of that of dysprosium ; there is a second activity of 
somewhat less intensity decaying with a period (2) of 4 h.; as lutecium 
(cassiopeium) and ytterbium are very closel^y related elements, and 
lutecium being usually contaminated with ytterbium, we considered it 
possible that the 4 h. period observed might be due to the presence of 
ytterbium in the sample investigated. A very pure lutecium (cassiopeium) 
preparation, however, prepared l)y Auer and kindly lent us by Prof. 
HoNiGSCHMiD. also showcd the 4 h. period. Furthermore the intensity 
of this radiation was stronger than that emitted by a pure ytterbium 
preparation activated by a neutron source of the same strength. So we 
must conclude that both the periods observed are due to lutecium. 
The long period of decay has not been observed by any experimenter 
besides us, presumably because the times of exposure have been too 
short. For the shorter period McLennan and Rann (5) give a value of 
3.6 h. and Rona (6) 4—5 h. The decay of the lutecium preparation lent 
us by Prof. Honigschmid is seen in Fig. 2. the time of exposure 
being 2.8 days. In comparing the intensities of the long and the short 
periods the former must be divided by 0.267 which value follows from 
a consideration of the relation J^ = '\ (1— c'^'M- where J^ is the 
saturation value of the activity. J^. the value obtained after t days, and 
P. the decay constant (= half-life/ln 2). As can be seen from the Fig. 
2b a third long period is present in the activated lutecium which is 
possibly due to the presence of small amounts of thulium. 



ABSORPTION OF SLOW NEUTRONS BY RARE EARTH ELEMENTS 
Determination cf the period of decay from absorption data 

When faced with the problem of determining the period of very slo^\ly 
decaying radioactive isotopes having half-lives of several months or 
years, decay measurements become very tedious. In such a case we can 
obtain information about the decay constant required by comparing 
the absorption of slow neutrons in the rare earth element in question 
with that in another rare earth element of known period. A knowledge 
of this ratio and of the activities obtained for Iwth elements after a known 



THE ACTION OF XEUTRONS OX THE RARE EARTH ELEMEXTS 55 

lime of exposure allows us lo calculaie the unknown period oi' decay 
provided we can assume that all the neutrons absorbed are captured 
by the nuclei of the absorbing element and that mainly thermal neutrons 
are involved in both cases. In the oxides investigated, only the nuclei 
of the rare earth element absorb, for oxygen nuclei capture only a small 
number of neutrons. Let us consid(M-, for example, the case of scandium. 
Denote by R^ the observed absorption ralio for equal numbers of scan- 
dium and dysprosium atoms, and by Rg the ratio of the activities obtained 

after an exposure of N days ; then the half-life of scandium is _L 

Rg 

days. We compared the activity of 66 mgm of scandium and 100 mgm 
of dysprosium and found after an activation of 24 days an activity 
ratio of 0.92x10" 2. During this activation time, full saturation of the 
dysprosium activity was obtained, while the scandium was far from being 
saturated. For equal numbers of scandium and dysprosium atoms we 
found an intensity ratio of 0.40 xlO"^. 

To compare the absorbing powers of scandium and dysprosium we 
inserted in the path of the neutron beam, which had been slowed down in 
the usual way by a block of paraffin, first, a layer of scandia (590 mgm/cm- 
Sc) and then a layer of dysprosia (340 mgm/cm^ Dy) and measured the 
activation of a rhodium foil in the absence and then in the presence of the 
absorbing layer. The amounts of the absorbing material necessary to 
reduce the activity of rhodium in each case to 90% of its initial value 
were calculated to be 300 mgm/cm^ Sc and 43 mgm/cm^ Dy. A more satis- 
factory way to proceed in comparing the absorbing powers would have 
been to have used a dysprosium indicator to measure the absorption 
in dysprosium and a scandium indicator to measure the absorption in 
scandium, but the small activation of scandium after a few days' expo- 
sure to neutrons rendered this infeasible. We have, however, applied 
the last mentioned method to compare the absorption of neutrons in 
dysprosium, europium, and holmium, as discussed in the next section. 
The comparison of the absorbing powers of equal numljers of atoms of 
dysprosium and scandium led to the result that the former absorbed 25 
times as strongly as the latter. It follows from this result and from 
the comparison of the activities of the two elements, that the half-life 
of2iSc is about tw^o months. A similar value was obtained by dtn-ay 
measurements. 

Strongly absorbing rare earth isotopes forming stable products 

The unusually strong activities of some rare-earth nuclei are to be 
ascribed to the existence of strong nuclear resonance levels in the nuclei 
in question, these levels corresponding to energies of slow neutrons 
abundant in the neutron beam passing through them, and also to the fact 



56 



ADVEXTURES IX RADIOISOTOPE RESEARCH 



Table 1. — Absorption of Slow Neutrons in Rare Earth Elements 
(Amount necessary to reduce the activity of the indicator by ten per cent) 



Element 


Indicator 


mgm/cm^ 


Europium. 

Dysprosium 

Holmium 


Europium 

Dysprosium 

Holmium 


13 

40 
120 



that the isotope formed by the capture process is not a stable one already 
known but an active one hitherto unknown. It is a matter of experience 
that a mass number cannot be occupied both by a stable and an active 
isotope of the same element, so that should the mass number 166 be 
occupied by a stable dysprosium isotope the high capturing power for 
slow neutrons shown by ^HDy would not lead to an active but to a 
stable dysprosium isotope. The appearance of a strong activity shows 
that at least one isotope of this element captures neutrons strongly, 
but high absorption does not necessarily imply strong activity because 
nuclei yielding stable isotopes can also be very strong absorbers of neu- 
trons. To obtain information about the existence of strongly capturing 
rare-earth nuclei not leading to the formation of radioactive products, 
we compared the activities of dysprosium, europium, and holmium with 
their absorbing powers for the same neutron beam as was used to activate 
them. The results of these measurements, in which the absorbing el- 
ement itself was used as indicator, are seen in Table 2. 



Table 2. — Absorption of Slow Neutrons in Rare Earth Elements 
( Amount necessary to reduce the activity of the indicator by ten per cent) 



Element Indicator 


mgm/cm" 


Europium 

Dysprosivim 

Holmium 


Rhodium 
Rhodium 
Rhodium 


16 
43 

160 


Gadolinium 
Samarium 
Yttrium 
Scandium 


Rhodium 
Rhodium 
Rhodium 
Rhodium 


2 

12 

500 

300 


Cadmium 


Rhodium 


18 



While the activity of europium is slightly smaller than that of dyspro- 
sium its absorbing power is more than twice as big ; europium thus 
absorbs slow neutrons to an appreciably larger extent than is to be 
expected from the activity of the radioactive europium isotope formed. 
To explain this discrepancy we have to assume that besides the 



THE ACTION OF >fEUTROJSfS ON THE RARE EARTH ELEMENTS 57 

9 h. period a second period, Jong and therefore not observed, is 
present. 

Samarium also shows an absorption stronger than is to be expec- 
ted from the activity of the known radiactive samarium isotope. 
Of the numerous isotopes of samarium not leading to tlic formation of 
active isotopes at least one must therefore have a strong resonance level 
for slow neutrons. In view of the fairly weak activity of samarium the 
absorption measurements could not be carried out by using a samarium 
indicator, so rhodium was used for that purpose. The results of these 
measurements and also of absorption measurements with other rare 
earths using rhodium as indicator are shown in ta1)le 3. 



Table 3. — Percentage of Initial Intensity of the Neutron 
Beam Present after the Passage of a "Thick" Layer 



Element mgm/cm* 


Intensities 


Samarium 

Gadolinium 

Dysprosium 


.580 
120 
610 


28% 
33% 
40% 


Cadmium 


390 


43% 



It is well-known that the activity obtained by the action of slow- 
neutrons is not a trustworthy measure of the intensity of the neutron 
beam, because the neutron absorbing powers of different elements are 
very specific and depend very much on the neutron velocities. The ambi- 
guity arising from this fact can, however, be avoided by using the same 
element as indicator and absorber in absorption experiments. Should 
that not be feasible, as would happen if, for example, the absorbing 
substance did not show any or had only a very slight activity — this is 
the case with gadolinium — it is advisable to adopt the following pro- 
cedure. The maximum absorption obtained in a thick layer of gadolinium 
is measured using, say, rhodium as indicator ; then the thick layer is 
replaced by a few milligrams of material and the absorbing power 
measured again. The first mentioned measurement gives the result that 
no more than 67% absorption can be obtained for the neutron beam 
in question through a thick layer of gadolinium, while the last mentioned 
measurement shows that2mgm of gadolinium are necessary to reduce the 
intensity of the neutron beam by 10%. To arrive at a figure giving the 
amount of gadolinium necessary to reduce the intensity of neutrons 
of such velocities as are actually absorbed in gadolinium we must mul- 
tiply 2 mgm by 0.67 and thus obtain a value of 1.3 mgm. The correspond- 
ing figures for a few elements are given in Table 4 and 5. Of all the rare 
earth elements gadolinium —as can be seen from the table — has the 



^8 



ADVENTURES IN RADIOISOTOPE RESEARCH 



Table 4. — Absorption of Slow Neutrons in Rare Earth Elements 
(Amount necessary to reduce the activity of the indicator Vjy ten per 
cent of that observed after pa.ssage of tlie neutrons througli a "thick" 

layer) 



Element 


Indicator 


mgm/cm- 


Samarium 

Gadoluiium 

Dysprosium 


Rhodium 
Rhodium 
Rhodium 


10 

1.6 
30 


Cadm^ium 


Rhodium 


12 



highest absorbing power ; it is indeed, as has already been shown b}' 
Dunning, Pegram, Fink, and Mitchell (8), the strongest known 
absorber of slow neutrons. In view of the very strong absorbing power 
of gadolinium great care must be taken in interpreting the results of 
absorption measurements on rare earth preparations which might con- 
tain traces of gadolinium. The presence of less than 1/2 per cent of 
gadolinium in erbium, for example, would suffice to explain the wdiole 
absorption shown by erbium. As europium is often contaminated with 
gadolinium we used various preparations of europium to compare the 
absorption in europium and dysprosium. One of the preparations was 
kindly given us by Prof. Prandtl and was entirely free of gadolinium ; 
it gave a value only slightly lower than the other specimens investi- 
gated. 

The high values found by different observers for the absorbing power 
of yttrium are clearly due to the presence of impurities in the prepar- 
ations used. According to Amaldi and his collegues (1) the absorbing 
power of yttrium is 70 per cent of that of cadmium, and Dunning, 
Pegram, Fink, and Mitchell (8) give 39 per cent ; whereas using very 
pure preparations as described on page 48 we find that yttrium is a very 

Table 5. — The Relative Activities of the Rare Earth Elements 



Element 
Bombarded 


Relative 
Intensit}' 


Element 
Bombarded 


Relative 
Intensity 


Yttrium 

Lanthanum .... 

Cerium 

Praeseodymium . 
Neodymium .... 

Samarium 

Europium 

Gadolinium 


0.5 
2 

4.5 

0.04 

0.6 

80 
very low 


Terbium .... 
Dysprosium . 
Holmium . . 
Erbium .... 
Thulium . . . 
Ytterbium . . 
Tjut,ecium . . . 




2.5 
100 
20 
0.35 
12 

0.25 
1.4 ; 1 







THE ACTION" OF NEITROXS OX THE KARE EAHTH ELK.MEXTS 59 

l)oor absorber, its absorbing power being only 4 per cent of that of 
cadmium and 0.3 per cent of that of gadolinium, if Ihe absorption of 
equal numbers of atoms of the different elements are compared. In 
Table 6 are given the relative intensities of the activities produced in the 
rare earth elements by neutrons that have been slowed down by large 
amounts of paraffin wax. We are still investigating the intensiti(>s 
obtained under the action of fast and semi-fast neutrons and the possible 
existence of resonance levels. 



Comparison belween ihe effect of neutrons on rare earlh elements and 
other elements 

As is shown in this paper numerous radioactive isotopes of the elements 
of the rare earth group are formed under the action of neutrons, a result 
which was to be expected from the known existence of a large number of 
stable isotopes of these elements. Thus the reactions of neutrons with 
the rare earth elements show the same typical features as their reactions 
with elements of lower and higher atomic number. The most remarkable 
feature is perhaps the comparatively frequent occurrence of pronounced 
resonance phenomena, which phenomena are much commoner among 
the rare earth elements than in any other part of the periodic system. 
This fact may be considered as a simple consequence of Bohr's 
theoretical considerations on neutron capture, since it would be 
expected that the distribution of resonance levels would be an especially 
close one in this region. In fact the product of the number of nuclear 
particles multiplied by the binding energy of a neutron in the nucleus 
reaches a maximum in the domain of the rare earth nuclei on accouni of 
the circumstance that Ihe Ijinding energy for higher particle numbers 
decreases considerably until — for the natural radioactive bodies — it 
has fallen to about half its maximum value. The more frequent occurrence 
of resonance capture in processes leading to the formation of stable 
isotopes, than in those giving radioactive isotopes is also in conformity 
with general experience and is easily explained by the theoretical con- 
siderations mentioned above since the distribution of levels will 
])e much closer in the former case on account of the fad that the binding 
"nergy is considerably larger in processes of this kind 1han in those 
leading to the production of unstable isotopes. 

The use of neutrons in analytical chemistry 

The usual chemical methods of analysis fail, as is well-known, for mosi 
<)[ the rare earth elements and have to })e replaced l)y spectroscopic, 
X-ray, or magnetic methods. These methods can now be supplemen- 



60 ADVENTURES IN RADIOISOTOPE RESEARCH 

ted by the application of neutrons to analytical problems by making 
use both of the artificial radioactivity and of the great absorbing power 
of some of the rare earth elements for slow neutrons. 

Qualitative analysis with the aid of artificial radioactivity is based 
on the determination of periods of decay. All rare earth elements have 
half-lives varying from a few minutes to a few month, so they can all 
be measured conveniently. The period of decay of 2.5 h., for example, is 
completely characteristic of dysprosium and is an unambiguous indication 
of its presence in the sample investigated ; as little as 0.1 mg can be 
determined without difficulty. We used the method of artificial radio- 
activity to determine the dysprosium content of yttrium preparations. 
The procedure was the following: we mixed 0.1%, 1% etc. of dyspro- 
sium with neodymium oxide, the latter being chosen because it is one 
of the cheapest rare earth elements, having a low neutron absorbing 
power as has yttrium, and determined the intensity obtained. The yttri- 
um sample to be investigated was then activated under exactly the same 
conditions, and a comparison of the dysprosium activities obtained 
gave 1% as the dysprosium content of the yttrium sample. 

Another very beautiful analytical method is based on the very different 
absorbing powers of the different rare earth elements. A sample, 5 mgm 
of which spread over 1 cm^ absorbed a quarter of the slow neutrons 
falling on it, could be identified at once as gadolinium, no other element 
having so high an absorbing power. 

Unlike the method of artificial radioactivity, the absorption method is 
limited in its application by the fact that the absorption measure is the 
sum of the absorptions of the different elements present in the sample. 
This limitation is, however, largely due to the fact that our knowledge of 
the absorption of neutrons and still more our devices for producing 
neutrons of different energies are only in an embryonic state. The absorb- 
ing powers of different nuclei depend to a high degree on the energy of 
the neutrons in question and the future development of our knowledge of 
neutron absorption will presumably make it possible to apply absorption 
methods of neutron analysis of great simplicity and reliability. This 
method of analysis, as also that based on periods of decay, gives a direct 
means of identification of the nuclei involved ; this distinguishes them 
from all other analytical methods, chemical, spectroscopic, X-ray, and 
magnetic, which are based on the investigation of the electronic proper- 
ties of the atom in question. 

Effect of neutrons on minerals containing rare earth elements 

Many of the rare earth minerals, because they are products of residual 
magmatic crystallisation, contain rare earth elements, thorium, and 
uranium, along with beryllium and other light elements. The las1 



THE ACTION OF NEUTRONS ON THE RARE EARTH ELEMENTS (51 

mentioned (>lement is I'ar the most effective neutron source under 
Jjombardnient with a-particles or with the y-rays emitted by uranium, 
thorium, and their disintegration products ; the nuclei of other elements, 
such as lithium, boron, magnesium, aluminium etc. are much less 
effective.* Jn minerals containing large amounts of strongly capturing 
rare earth elements, the neutrons produced in the mineral or in its 
surroundings are absorbed to a large extent in the element in question. 
The mineral gadolinite, for example, contains about 50% of rare earths, 
of which according to Goldschmidt and Thomassen** up to about 
15% is gadolina ; this mineral often contains, too, other light elements 
including considerable amounts of beryllium, about 0.3% of thorium, 
and some uranium as well. 1 gm of thorium and its disintegration pro- 
ducts produces up to 10^ neutrons per year or in all 10^^ neutrons since 
the formation of the minerals. If these neutrons are all absorbed in 
1 kgm of the mineral in question and are absorbed primarily by the gado- 
linium content, lO^^ gadoKnium atoms will be formed having an atomic 
weight one unit higher than before the absorption. As 1 kgm gadolinite 
contains about 10-'^ of gadolinium atoms the equivalent weight of gadoli- 
nium will increase during that long span of time by but one unit in 
the fourth decimal place. 

While this result is only a very rough estimate it suffices to demonstrate 
that some of the rare earth elements which primarily form higher stable 
isotopes by capturing neutrons, increase in equivalent weight as time 
proceeds. Dysprosium on the other hand when decaying forms holmium, 
holmium forms erbium etc.; the process in such cases leads to an increase in 
theamountsof rare earths of higher atomic number and to a correspond- 
ing decrease in the amounts of those of lower atomic number. Such 
behaviour is not confined to the rare earth elements ; during their 
presence in the earth's crust many elements heavier than zinc will 
undergo increases, though small ones, of their equivalent weights oi 
of their abundance relative to the lighter elements. The first named 
behaviour is shown primarily by even and the last named by odd ele- 
ments, because elements having an odd atomic number have always a 
few isotopes only so that the consecutive mass numbers are not filled by 
stable isotopes and the formation of radioactive isotopes through neutron 
addition is possible. In the case of several even elements like cadmium, 
tin, gadolinium, osmium, mercury, lead etc., a long series of consecutive 
mass numbers are filled by stable isotopes so that the capture of neutrons 



* \\'o compared the activities obtained when dysprosia \\as bombarded with 
iH'viiions of a bciylhum-radon and a magnesium-radon source in the presence 
of large amounts of paraffin wax and the figures obtained were as 100 : 1. 

** V. M. Goldschmidt and L. Thomassen, Oslo Viclskcip Scl-skujJets Skrifter I, 
Xr. 5, S. 44 (1924). 



62 ADVENTURES IX RADIOISOTOPE RESEARCH 

leads chiefly to the formation of higher stable isotopes. It is therefore 
even elements that undergo an increase of their equivalent weights 
with time while the relative abundance of the elements of odd atomic 
number shifts towards the heavier elements. 

Below zinc, conditions are very different : the result of neutron action 
in minerals leads here often to the formation of elements of lower 
atomic number and only to a smaller extent to the formation of 
heavier isotopes or heavier elements. For example, bombardment 
of aluminium leads to the formation of a magnesium isotope and to 
a sodium isotope ; the branching ratio between these two processes 
depends greatly on the energy of the neutrons. 



Sunimary 

The artificial ladioactivity of the rare earth elements including scandium 
and yttrium was investigated. The periods of decay of numerous radioactive 
isotopes produced lie between 5 min. and a few month . The biggest and smallest 
saturation intensities of the radiation emitted by these isotopes are in the ratio 
10,000 : 1. The half- value thickness in aluminium of the (^-radiation emitted 
was measured in several cases, and, in some cases, the maximum energy of the 
continuous ^-ray spectrum and Fermi's constant a as well. 

The absorption of slow neutrons in rare earth elements was measured with 
a view to disco\ering the presence of strongly absorbing nuclei not giving rise 
to active isotopes. 

The application of artificial radioactivity to analytical chemistry is discussed. 

It is shown that the combination weight of the rare earth elements occurring 
in minerals in which a continual production of neutrons takes place has undergone 
a slight change during geological time. 



References 

1. E. AMAI.DI, E. Fermi et al., Proc Roy. Soc A. 149, 522 (1935). 

2. G. Hevesy and Hilde Levi, Nature 136, 103 (1935) and Nature 137, 849 
(1935). G. Hevesy, Nature 135, 96 (1935); Roy. Danish Acad. (Math.-fys. 
Medd.) XIII, 3, (1935). 

3. S. SuGDEN, Nature 135, 469 (1935). 

4. J. K. Marsh and S. Sugden, Nature 136, 102 (1935). 

5. I. C. McLennan and W. H. Rann, Nature 136, 831 (1935). 

6. E. Bona, Wiener Akad. Anzeiger 27, (1935) and 73, 159, (1936). 

7. J. C. Chadwick and M. Goldhaber, Camb. Phil. Soc. 31, 612, (1935). 

8. J. R. Dunning, G. B. Pegram, G. A. Fink and D. P. Mitchell, Phtjs. 
Rev. 48, 265, (1935). 

9. N. Bohr, Nature 137, 344 (1936). 

10. H. A. Bethe, Phys. Rev. 50, 332 (193G). 



Oiiginally published in tho Kgl. Danske Videnskabei'nes Selfikab. Mathemcttisk- 

fijsiskc Meddeldser. 15, II (1938) 

5. ARTIFICIAL ACTIVITY OF HAFNIUM AND 
SOME OTHER ELEMENTS 

G. Hevesy and Hilde Levi 
From Tho Institute of Theoretical Physios, University of Copenhagen 

ARTIFICIAL RADIOACTIVITY OF HAFNIUM 

Some time ago we found that under the action of neutron bombardnienl 
a radioactive isotope of hafnium is produced, the activity decaying 
with a period of a few months (4). To determine the period of decay 
more exactly, we activated 280 mgni of hafnium oxide prepared by 
one of us (3) by placing it in a paraflin block together with radium- 
beryllium sources, containing 600 mgm of radium element as sulphate 
and twenty times as much metallic beryllium powder. After irradiation 
for three months the hafnium oxide was removed from the paraffin 
block and put into an aluminium dish having a surface of 1.2 cm^ and 
a height of 2 mm. The dish was placed directly below the aluminium 
window of our counter, the window having a thickness of about 20 i-i. 
We followed the decay of the hafnium preparation for 200 days by 
comparing its activity with that of an uranium standard. The decay 
curve obtained is seen in Fig. 1 and Table 1. 

From the latter we can conclude that the half life of liafnium is 
55 + 10 days (standard mean square deviation). Our initial activity 
was 20 counts per min, the natural effect being about 4 counts per min. 
We followed the decay curve until we had a net activity of 1 counts per 
min. From the fact that, in spite of the long activation, such a modest 

Table 1. — Dec.w-measueement of Hafnii'm 



Date Nr. of Days 

1 


Counts/min 


17. VIII. 36 

18. VIII. 36 

24. VIII. 36 

12. IX. 36 

29. X. 36 

7. I. 37 

22. I. 37 

2.5. II. 37 




1 

7 

25 

73 

143 

1.5S 

192 


16.1 
15.3 
13.7 
12.0 

7.8 
2.3 
1.8 



64 



ADVENTURES IN RADIOISOTOPE RESEARCH 



activity was obtained we can conclude that hafnium does not belong 
to the elements showing a strong artificial radioactivity. This is partly 
due to the fact that the capture of neutrons by most of the hafnium 
isotopes leads, as explained later, to the formation of a heavier stable 




200 days 



F"ig. 1. Decay Curve of Hafnium. 



isotope ; as stable isotopes 176, 177, 178, 179, and 180 are known and 
only the absorption of neutrons by the last mentioned isotope can lead 
to the formation of an active product. The relative abundance of the 
isotopes in the naturally occurring element hafnium, as determined 
by Aston, is seen from Table 2. 

Table 2. — Relative Abvxdance of the Hafnium Isotopes 



Mass number 


Aljundance 


176 


5% 


177 


19% 


178 


28% 


179 


18% 


180 


30% 



ARTIFICIAL ACTIVITY OF HAFNIT'M AND SOME OTHEK ELEMENTS (if) 

We measured also tlic absorption in aluminiuui of the /?-rays emitted 
by hafnium. The values obtained ai(> seen from Table 3. 

Tablk ."}. — Absorption in Aluminium of the j6-k.\vs Emittku 

BY Hafniltm 



Thickness of tlie Al-£oil j Uou uts/miii. 




1 1 mgm/cm^ 
16.5 mgm/cm^ 



1.5.8 

10.2 

7.4 



(half value thickness : 16 i 1 mgm/cm^). 

From the figures in Table 3 follows that an aluminium layer of 16 
mgm per cm- reduces the intensity of the ^-rays emitted by a hafniinn 
oxide layer of 230 mgm/cm^ to one half of its initial value. The com- 
parison of the absorbing power of aluminium for the /5-rays of hafnium 
and scandium, decaying with periods of 55 and 90 days respectively, 
shows no great difference ; the ratio of the two half- value thicknesses 
l)eing 1.2. The softness of the hafnium radiation is partly responsible 
for the low activities ol)tained after long exposure of hafnium oxide 
with radium-beryllium sources of a few hundred millicurie, the /5- 
ladiation emitted being absorbed to an appreciable extent in the haf- 
nium oxide sample itself. In the case of hafnium, as already mentioned, 
every place between the mass numbers 176 and 180 is occupied by a 
known stable isotope ; the formation of the active hafnium isotope 
is presumably due to the process 

vaHf-f on = 72Hf. 

On emitting /^-rays according to the equation 

'?^Hf = mi\ -f (^ 

the active hafnium isotope becomes the only stable isotope of tantalum 
known. Hafnium is thus partly converted into tantalum under the 
action of neutron bombardment, while, as shown by us previously, 
hafnium is formed under the action of neutrons on lutecium. It is quite 
possible that, under bombardment with a powerful stream of deuterium 
or of neutrons, further decay periods of hafnium will be discovered. 

THE EFFECT OF NEUTRON BOMBARDMENT ON SCANDIUM 

A few years ago we embarked on the investigationof the effect of neut- 
ron bombardment on scandium, (4), (5), (6), chiefly in the hope of being 
able to prepare an artificial radioactive isotope of potassium and to ob- 

O llevesy 



66 



ADVEXTURES IX RADIOISOTOPE RESEARCH 



tain some information on the then not entirely elucidated nature of the 
natural radioactivity of potassium. We bombarded a few grams of very 
pure scandium oxide prepared by Prof. Sterba-Bohm and used by Prof. 
HoNiGSCHMiDT in his work on the atomic weight of scandium. After 
neutron bombardment the scandium oxide was dissolved in dilute hydro- 
chloric acid and 100 — 150 mgm of sodium chloride as a carrier of ^^K 
and the same amount of calcium oxide was added. The filtrate obtained 
after precipitation with carbonate-free ammonia was treated with oxalic 




^ 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 36 40 42 days 

Fig. 2. Dec;iy Curve of the Potassium Precipitate. 



acid and the calcium oxalate formed was removed. The sodium chloride 
which had been added to the solution of the scandium chloride compound 
was recovered after the removal of the ammonium chloride content of 
the last filtrate by evaporation. The activities of the three fractions, 
namely scandium oxide, sodium chloride, and calcium oxalate, were 
then determined. Only the two first preparations mentioned were found 
to be active. The activity of the scandium oxide decayed very slowly 
while the various sodium chloride fractions obtained in different ex- 
periments lost half of their slight activity within 10 and 18 hours. We had 
just finished the experiment mentioned when a note was published by 
Fermi and his collaborators (1) concerning the action of neutrons on 
potassium. They found that potassium captured neutrons by giving 
birth to a potassium isotope decaying with a half- life of 16 hours. The 
values found by us for the period of the slight activity of different 
potassium preparations obtained from irradiated scandium showed a 
half- life between 10 and 18 hours; we thought it justifiable, therefore, 
to identify the element found by us with that found by Fermi and his 
collaborators. The initial activities measured amounted usually to about 
10 counts/min. In one case, through the courtesy of the Medical Radi- 
um Station and Dr. J. C. Jacobsen, we obtained an unusually strong 
neutron source containing 600 millicuries radium-emanation. The decay 
curve obtained for potassium 42 in this experiment is seen from Fig. 2. 



ARTIFICIAL ACTIVITY OF HAFNIUM AM) SOME OTHKK ELEMENTS 67 

The activity found by us in the filtrate of scandium precipitate couhl 
only be that of ^^K, as the presence of active impurities was excluded 
by the fact that the above mentioned very pure scandium sample was 
used. The possibility that we measured the half- life of radioactive 
sodium of 15 hours can be excluded with certainty not only for- the 
reason mentioned above but also for the following reasons : Sodium, 
iiNa, can be prepared either from '-^Na by simple n(>utron capture, or 
from magnesium if the capture is followed by emission of a proton, 
or from aluminium if the capture is followed by emission of ana-particle. 
From the first mentioned process with the neutron sources at our disposal 
only very weak activities can be obtained even when starting with pure 
sodium. To prepare measurable amounts of radio-sodium from a few 
grams of impure scandium oxide an appreciable amount of magnesium 
or aluminium would have had to l)e present in the preparation. 15 mgm 
of aluminium mixed with 150 mgm of ammonium nitrate, for example, 
gave after activation to saturation less than 0.5 counts/min. and the 
activity obtained by similar amounts of magnesium with the same sources 
as used when activating scandium was still smaller. The amount of 
radio-sodium obtained from magnesium is less, and that of sodium by 
neutron capture very appreciably less than that obtained from alu- 
minium. Scandium having just one stable isotope liSc, only the potassium 
isotope ^^K can be produced under neutron bombardment according 
to the equation 

MSc + Sn = liK-f|He. 

In the case of neutron capture by potassium, on the other hand 
both reactions ^^K + n and ^^K -|- n can occur. While Fermi and his 
collaborators left it open which of the two last named potassium iso- 
topes were produced, we could conclude from our experiments that the 
process witnessed by Fermi was ^^K -f- n = ^^K, and also that the 
process ^^K very probably leads to the formation of the potassium 
isotope *°K which is responsible for the natural radioactivity of potassium. 
Recently, Walke (9), by making use of Lawrence's powerful cyclotron, 
which supplies a many thousand times stronger neutron beam as ob- 
tained from our radium-beryllium sources, was able to follow the decay 
of ^2Jy through ten periods and determined its half life period to be 
12.4 7^7 0.2 hours, i. e., a somewhat lower value than that following 
from the investigations of Fermi and from our Fig. 2. 

Besides preparing ^^K according to the equation 

If Sc + in = IfK 4- a . 
we succeeded (4) also in preparing this isotope by the process 

42/ 
20*^ 



^^Ca + Jn = ?,K + m 



6* 



68 



ADVENTURES IN RADIOISOTOPE RESEARCH 



Walke (10) while reproducing Fermi's results and also ours as to the 
preparation of ^^K from scandium, was unable to reproduce our ex- 
periments in which ^"-^K was prepared from calcium. This negative result 
induced us to repeate our experiments, this time by bombarding with 
fast neutrons as much as 1 kgm of calcium carbonate. These were dissolved 
in a minimum amount of HCl, precipitated by a minimum amount of 
ammonium oxalate, which sufficed to precipitate all calcium after 
dissolving 100 mgm of sodium chloride as carrier. Before we finished 
these experiments, a second paper of Walke (12), (13), was publis- 
hed in which he describes succesful experiments in producing ^'-K 
from calcium, thus corroborating our statement. 



ACTIVITY OF SCANDIUM 

After the removal of the radio-potassium produced, the scandium 
was still showing a weak activity which could not be removed by 
chemical operations and which is possibly due to a radioactive isotope 
of scandium. The decay of the weak activity of scandium observed for 
240 days is seen from Table 4, which shows the presence of a very 
weak activity decaying with a period longer than a year. We could 
not follow up this very weak activity further but concentrated our inte- 
rest on another period obtained after activating for 24 days in a 
paraffin block which contained emanation-beryllium sources of an 
average strength of 50 millicuries. The result obtained is seen from 
Fig. 3a ; the half- life works out to be 90 i 5 days. 



Table 4. — Activity of a Scandium .Samplk aktkr Removal of 

Potassium 



Date 


Xr. (if Diij'S 


Counts/mill 


5. III. 36 

21. III. 36 

24. IV. 36 

20. V. 36 

5. VI. 36 

1. VII. 36 

13. VIII. 36 

24. X. 36 




16 

50 

76 

92 

117 

161 

233 


7.3 + 0.6 

6.0 + 0.4 

7.2 + 0.4 

5.1 + 0.4 
5.7 + 0.4 
4.9 + 0.3 

5.3 + 0.4 

4.4 + 0.3 



In the nexl sv\ of experiments we activated simultaneously three 
scandium preparations for 50 days with radium -])eryllium sources of 
a strength of about 200 millicuries : one in tli(> usual way inside the 



ARTIFICIAL ACTIVITY Oi' JIAIWII'M AM) SOMK OTllKK ELEMENTS 



69 



paraffin block, 1 Ik^ second one in a paraffin l)iock but Avil h 1 he preparation 
surrounded by a shield of cadmium, which al)Sorbed nearly 100% of 
the C-neutrons, and th(> 1hird one with fast neutrons. The result of the 
activation of the first named sample is seen from Fi^. 3b. The investig- 
ation of the second sample led to the result thai in the presence of 




~i I 1 1 r 

20 40 60 80 100 



200 days 



Fii^. 3. Dot-ny Curve of .Scandium InadiatL-d in a Paraffin Block for 

a) 24 Dav.s h) .'>() Davs. . 



cadmium the artificial radioactivity of scandium is reduced to 2% of 
the value obtained in the absence of cadmium. From Fig. 3 there 
follows for the half-life period of scandium the value 90 zb 5 days. 
Quite recently Walke (11), by making use of Lawrence's powerful cyclo- 
tron, bombarded scandium with deuterons and obtained a period of 
85 i 2 days. 

We want furlhermorci to mention an early experim(>nt in which we 
bombarded scandium with fast neutrons emitted by a mixture ol 
600 millicuries emanation and beryllium powder ; we observed a period 
of decay of about 50 hours. As shown by Pool, Cork, and Thornton (8). 



70 



ADVENTURES IX RADIOISOTOPE RESEARCH 



and by Walke( 12), under bombardment with fast neutrons the following 
two reactions occur as well : 

MSc+5n = MSc + 3^n 
ifSc + in = llSc + 2 In 

the scandium isotopes obtained emit positrons and have half- lives of 
4 and 43 hours respectively ; it was presumably the last mentioned 
reaction which we observed. 

Scandium 44 was also produced (13) by the action of a-particles on potas- 
sium 41 and (11) by the action of deuterons on calcium 43, w hile scandium 43 
was produced (1 1) by the action of a-particles on calcium 40, and (2) by the 
action of deuterons on calcium 42. 

Walke was furthermore successful in producing scandium 42 under 
the action of a-particles on potassium 39, and of scandium 41 under the 
action of deuterons on calcium. The list of the known radioactive iso- 
topes of scandium is seen from Table 5. 



Table 5. — • Active Isotopes of Scandium 
(According to Walke [11]) 



Active Isotope 


Particle Emitted 


Half-life 


"Sc 


positron 






53 min. 


*2Sc 


positron 






4.1 h 


"So 


positron 






4.0 h 


"So 


positron 






52 h 


*«Sc 


electron 






85 days 






and 


possibly also 






a 


period of about 










1 year. 



We measured the reduction of counts when covering an active scandium 
preparation decaying with a period of 90 days with aluminium foils 
of varying thickness. The result obtained can be seen from Table 6. 

Table f>. — Absorption in Aluminium of the /S-rays 
Emitted by Scandium 



Thickness o 


the Al-foil 


Couuts/inin 






o..") mgm/cm^ 


12.5 


P^ experiment 


s.s 




1 1 .0 mgm/cm^ 


7.1 







Hi. 8 


2nci experiment 


11.0 mgm/cm^ 


35.3 




16.5 mgm/cm^ 


25.8 



(half- \aluc thic-kncss : 13 ± 1 mgm/cm'') 



ARTIFICIAL ACTIVITY OK HAFNIUM AND SOME OTHER ELEMENTS 71 

In view of 1 he softness of 1 he /:?-rays emilled we used tliin scan- 
dium oxide layers ; about 50 nigm/cm'^. In spite of the thin layers 
used the soft components were absorbed in the preparation to an appreci- 
ably greater extent than were the hard ones ; accordingly we have to 
leckon with the possibility that the radiation emitted by scandium 
is still softer than indicated by the figures of Table 6. 



THE RADIOACTIVITY OF EUROPIUM AND ITS ANALYTICAL 

APPLICATION 

In their fundamental research on the action of neutrons Fermi and 
his collaborators (1) investigated also the activity of a gadolinium pre- 
paration bombarded by neutrons and found an activity decaying with 
a period of 8 hours. Afewyearslater,SuGDEN (7), investigating the radio- 
activity of europium, discovered a very strong activity decaying with 
a period of 9.2 h. and, at that time, interpreted the above mentioned 
period of decay of gadolinium to be due to the presence of some europium 
in the sample investigated. Investigations carried out by us, in whicli 
we made use of different gadolinium samples prepared by Prof. Rolla 
and partly by Prof. Prandtl and the late Baron Auer v. Welsbach, 
confermed completely the conclusion arrived at by Sugden, and this 
induced us to make use of the radioactivity of europium produced under 
the action of neutrons to determine the amount of europium present 
in gadolinium preparations. Prof. Rolla, being engaged in the prepara- 
tion of large amounts of pure gadolinium compounds, sent us several 
samples, the europium content of which he wished ascertained. We 
describe in the following the analytical procedure used by us. 

Thin layers of the gadolinium oxide samples to be investigated were 
fixed between two glass plates and placed within a paraffin block. 
Usually we investigated simultaneously the activation of 4 symmetrically 
placed preparations. It is of importance to bombard layers having the 
same thickness anrl to bombard them with neutrons in such a way that 
each preparation is hit by the same number of neutrons ; the latter 
was achieved by arranging the sources in the block circularly. We used 
in these experiments radium-beryllium sources containing 600 mgm 
of radium, the neutron emission of which corresponds to that from about 
400 millicuries of radium emanation ; in addition a beryllium-emanation 
mixture containing 300 millicuries emanation was also present. After 
irradiating the samples for 3 days they were homoginized and each 
sample placed in a small aluminium dish having a surface of 1.2 cm- 
and put below the window of a Geiger-counter. The intensity of the 
activity of the different gadolinium samples investigated is proportio- 
nal to their europium content. In order to arrive at a figure stating 



72 ADVEXTTRES IX RADIOISOTOPE RESEARCH 

the europium concentration, we added 2% of europium oxide to a puie 
gadolinium preparation denoted as standard sample in Table 7 and 
compared the activity of the latter with that of the gadolinium prepara- 
tions of unknown europium content. The results are seen from Table 7. 



Table 7. — Activity of Different Gadolinium Preparations 
(The sample labelled "Standard" is the GdjOa to wliich 2% 'EiuO^ was 
added: samples 1 — 4 represent progressiv stages in the purification process 

can-ied out by Prof. Rolla) 



Samples 


couuts/min 


"Standard" 


125 


1 


60 


2 


60 


3 


30 


4 


25 



That, in spite of the large amount of radium and emanation used, the 
activities measured were not stronger is partly due to the high absorbing 
power of gadolinium, which reduces the density of thermal neutrons. 
This effect is especially marked on account of the fact that the thermal 
neutrons diffuse and are likely to pass through the preparation several 
times. The latter effect can be best estimated by comparing the activity 
of pure europium oxide with that obtained when this material is 
embedded in gadolinium oxide. We activated simultaneously 200 
mgm of europium oxide and 200 mgm of gadolinium oxide containing 
2% of europium. If gadolinium absorbed to the same extent as 
europium, the first named preparation should be 50 times more active 
than the last mentioned one. Actually we find the ratio to be 200 from 
which it follows that the presence of gadolinium in our preparations 
reduced the activity of europium to y^ of the value which would have 
been obtained if the same amount of europium oxide had been subject- 
ed to irradiation. 

Suniinary 

The irradiation of liafiiium with noutrons has been shown <o produce a radio- 
activity with a half-hfe of 55 ± 7 days which may be ascribed to ^^g^lf- The 
intensity of the fi-rays emitted is reduced to half of its initial value by an alumi- 
nium foil having a weight of 16 mgm/cm^. 

Scandium, ^JSc, was found to decay with a half-life of 90 ± 5 days. The half 
^•alue thickness for the absorption in aluminium of the /?-rays from this element 
was found to be 13 mgm/cm^. 

The euiopium content of gadolinium oxide samples prepared by Professor 
LuiGi RoLLA was determined by making use of the artificial radioactivity- pro- 
duced imder the act ion of neutrons on the europium present in his samples. 



ARTIFICIAL ACTIVriV OF HAFXIV.M AM) SOME OTH Kl( KhK.ME.NTS Tli 

References 

I.E. Amaldi, E. Fkk^ji and otlicrs, Proc. Uoy. Sac. A 149, -322 (1935). 

2. O. R. Frisch. Xdliire 136, 220 (1935). 

3. G. Hevesy, Kgl. Danske Vid. Selsk. Math.-fijs. Medd. 6, 7. S. 91 (1925). 

4. G. Hevesy and IIilde Levi, Natiivc 135, 580 (1935). 

5. G. Hevesv, KgJ. Danske Vid. Selk,s. Math.-fys. Medd. 13, 3 (J935). 

(). G. Hevesy aiul Hilde Levi, Kgl. Danske Vid. Seh-k. Math.-fys. Medd. 14, 

5 (1936). 
7. S. SiTGDEX, Nature 135, 469 (1935). 

S. M. L. Pool, J. M. Cork and R. L. Thornton, Fhys. Ber. 52, 41 (J 937). 
9. II. Walke, Fhys. Rev. 51, 439 (1937). 

10. D. G. Hurst and H. Walke, Fhys. Rev. 51, 1033 (1937). 

11. H. Walke, Fhys. Rev. 52, 400 (1937). 

12. H. Walke, Fhys. Rev. 52, 663-669 (1937). 

13. M. Zyw, Xatiire 134, 64 (1934). 



74 ADVENTURES IN RADIOISOTOPE RESEARCH 



Comment on papers 4 and 5 

Usually the radioactive indicator must be added to the element the atoms of 
which are to be traced. It is, however, also possible to produce the radioactive 
tracer in situ by bombarding the sample with a neutron stream or other energy- 
rich radiation. 

In contiast to present days very few people disposed of rare-earth elements 
before \^'orld War II. Among those was my friend Professor Luigi Rolla, 
33rofessor at the University of Florence. We used to analyse his samples by 
making use of the method of X-rays analysis described in the comment to 
papers 3 and 15. After preparing a few kilograms of gadolinium oxide he 
wished to find out whether or not his samples were free from EujOg, the most 
likely impurity present in Gd203. At that date we had no X-raj' spectrograph at 
our disposal and in order not to disappoint Professor Rolla we tried t6 answer 
the above question by exposing 50 mgm of his sample in a paraffin block to the 
effect of neutrons emitted by a mixture of 600 mgm radium and beryllium 
and 300 ]\Ic of radon and beryllium. Neutron sources were placed in the 
paraffin block to obtain slow neutrons which are strongly captured by europium 
producing a radioactive europium isotope. They are strongly absorbed by gadoli- 
nium as well, their absorption leading, however, to the production of stable 
gadolinium isotopes and not to a radioactive isotope of this element ; the latter can 
only be produced by more intense neutron streams than applied in our expcr-iment . 
The presence of an activity in RoUa's exposed samples decaying with a half-time 
period of 9.2 hr indicated the presence of some europium in his preparations. 
To carry out a quantitative analysis we added to a known amount of pure Gd203 
(obtained from the great rare-earth chemist Auer von Welsbach) known amounts 
of pure EujOg (also obtained from him). The comparison of the activity of RoUa's 
samples with those of these standard preparations lead to the result that Rolla's 
purest gadolinium oxide sample contained 0.40, his least pure sample 0.96 per 
cent of europium oxide. 

We had already previously, as described in paper 4, applied this method of 
activation analysis in the determination of dysprosimn present inyttrivrm samples. 
The determination of europiuni in gadolinium is unsurpassed in its simplicity 
and sensitivity. Europium being the element which can be determined with 
the greatest sensitivity by activation analysis. We A\ere thus forttrnate to be 
faced with the task of applying this newly introduced method in a case 
which proved later to be the most favourable one. The modest neutron flux 
emitted by our radium-beryllium sources allowed not less than 0.01 per cent 
of europium to be determined. By making use of the neutron flux of the cyclotrorr 
Seaborg and Livingwood could determine 6 p. p. m. of gadolinium in iron by 
activation analysis and after the availability of pile-emitted netrtrons of great 
density such small amounts of europium could be determined as 10^^* mgm 
In the determination of gadolinirrm we availed ourselves of the very high absorb- 
rng power of tliis element for slow neutrons, thus of an absorbtion method. 

References 

Seaborg arrd LivrNGWOOD (1938) J. Awev. C'hcm. Soc. 60, 1784. 



Originally publishod in Phys. Z. 15, 797 (1914) 



6. THE PROBLEM OF THE ISOTOPIC ELEMENTS 

G. IIevesy and F. Paneth 
From the Institute of Radium Reseaich of the N'^ionna Academy of Science 

1. THE ISOTOPE CONCEPT 

It is well known that the separation methods of analytical chemistry 
have failed when dealing with some radioelements : Nobody has ever 
succeeded in separating radium-D from lead, mesothorium from radium, 
or ionium from thorium, nor has it once been possible in these and nu- 
merous other cases to achieve even a slight enrichment. As more un- 
successful experiments became known, the workers in this field adopted 
the view that they were concerned with an inseparability of quite a 
different kind from that operative with, for example, the rare earths. 
F. SoDDY^ was the first to give clear expression to this view by desig- 
nating such elements as "chemically and physically practically iden- 
tical" and also to search systematically for new examples of such in- 
separability among the radioelements^. 

Especially striking in connexion with the inseparable elements was 
tiie fact that they frequently have considerably different atomic weights 
which, since the a-particle was known to be identical with the helium 
atom, could be calculated in many instances with certainty ; for exam- 
ple, the end product of the uranium series, radium-G, which is generally 
regarded as lead, must have an atomic weight different from thai of 
ordinary lead^. Confirmation of the correctness of this conclusion has 
been obtainerl from the recently performed determinations of atomic 
weights*, which demonstrated that the lead from pitchblende has in 
fact an appreciably lower atomic weight than ordinary lead and the 
lead from thorium minerals. 

1 F. SoDDY, J. Chem. Soc. 99, 72 (1911). 

2 A. Fleck, J. Chem. Soc. 103, 381 (1913). 

3 See, for example, G. Hevesy, Phys. Z. 14, Ul (1913) ; F. Soddy, J. Chem. 
Soc. 105, 1402 (1914). 

* AI. Lembert, see K. Fajans, Z. Elektrochem., 1 June (1914) who suggested 
those experiments: O. Honigschmid, Ibid.; M. Curie, C. R. Acad. Sci., Paris 
•June (1914). 



76 ADVENTURES IN RADIOISOTOPE RESEARCH 

The question of the identity of different elements was given increased 
attention since this was the basis of arranging the radioelements in the 
periodic system^. K. Fajans^ has indicated that this idea can be carried 
throughout the periodic system and that the ordinary elements also 
are probably mixtures. Fajans has given the tame "pleiade" to such 
a group of elements which occupy the same position in the periodic 
system ; the separate members were called "isotopic'' elements by 
SoDDY. The lack of an ionium spectrum in ionium-thorium samples^ 
could scarcely be explained on any assumption other lhan that the 
isotopic elements show no differences in then- spectra. 

The theory of isotopic elements was not readily acceptable to chemists 
and physicists ; to the former, because ever since the formulation of 
the periodic system thej^ had been accustomed to regard the atomic 
weight as a fundamental property of an element ; to the latter, because 
there was no known instance in which two different elements exhibited 
the same spectrum and such a hypothesis seemed difficult to unify with 
the prevailing ideas on the origin of spectrum lines^. These doubts were 
removed and the whole concept of the nature of isotopic elements was 
simultaneously given considerably more weight by the ideas, developed 
by E. Rutherford^ and N. Bohr^, on the constitution of the atom, 
and by the experiments of Moseley'^ on the X-ray spectra of the ele- 
ments. According to the Rutherford model of the atom, the mass of the 
atom is associated with an extremely small volume at the positively 
charged centre and the number of positive charges, and not the atomic 
weight, is primarily responsible for the properties of the corresponding 
element. Since the number of electrons Avhich occupy the volume bet- 
ween the nucleus and surface of the atom is given by the size of charge 
on the positive nucleus and all chemical and physical properties of the 
element depend on the number and arrangement of these electrons ; 
gravitation and radioactivity are excepted. Instability of the nucleus 
results in radioactive phenomena and the fact that the nuclei of two 
atoms have the same charge and the same physical and chemical pro- 
perties but different mass and stability (e. g. radium -D and lead) agrees 
very well witli Ihe Rutherford-Bohr theory. 



1 A. S. Russell, Chem. News 107, 49 (1913) : K. Fa-t^^ns, Phijs. Z. 14. 13r, 
(1913); F. SoDDY, Chcm. Netcs 107, 97 (1913). 
2K. Fajans, Chem. Ber. 46, 422 (1913). 

3 F. EXNER and E. Haschek, Sitz. Ber. Akad. Wiss. Wienl2\, 175 (1912); A. S. 
Russell and R. Rossi, Proc. Roy. Soc. 87, 478 (1912). 

4 A. Schuster, Nature 91, 30 (1913). 

5E. Rutherford, FMl. Mag. 21, 669 (1911). 

«N. Bohr, Fhil. Mag. 26, 1 (1913). 

7 IT. Moseley, Phil. Mag. 26, 1024 (1913). 



THK I'KOIJI.KM OF IIIK ISOTOPIC ELEMENTS 77 

Determination of Ihe charge on ihe nucleus can be made approxima- 
lel}^ as a result of Ihe experiments on scattering of a-particles by M. 
(Ieiger and E. Marsdex, and more accurately by the recently perfor- 
med study by H. Moseley on X-ray spectra. A knowledge of the wave- 
length of the characteristic X-radiation of an elemenl permits calcu- 
lation of th(> nuclear charge when certain assumptions are made ; it 
was thus found that this charge always increases by unity on moving 
from one position in the periodic system to the next higher^. Generally 
1 his means a climb to the element with the next higher atomic weight 
l)Ul. in a few exceptional cases, where the chemical properties force 
t he element with the lower atomic weight to be arranged higher in the 
system (e. g. cobalt and nickel), the rule stated above still applies and 
t hus demonstrates that the number of charges, and not the atomic weight, 
determines the position of an element in the periodic system. According- 
ly, the separate positions can be numbered by stating the nuclear charge ; 
aluminium, for example, thus acquires the atomic numlier 13, gold 79, 
etc.. and between these all the available numbers except three are already 
representative of known elements. E. Rutherford and C. Andrade- 
have proved directly, by determining the X-ray spectrum of radium-B. 
which was found to be the same as that of lead, that there are ele- 
ments having different atomic weight but the same nuclear charge. 

Isotopic elements differ, according to this observation, only in the 
structure and mass of the nucleus. The structure does not enter into 
the ordinary physics and chemistry but is only of importance to the 
radioactivity. The ladioactive properties, how'ever, were the chief 
means of differentiating the isotopic element and, with a few exceptions 
(metaneon, the different kinds of lead), even now we are only aware 
of the existence of such isotopic elements in those examples in which 
at least one of them is radioactive. Separation by utilizing the radio- 
active differences does not seem to be conceivable ; it is otherwise with 
the second fundamental property of the nucleus, gravitation, which 
should permit both distinguishing and separating. 

It is useful in these discussions to distinguish between the gravita- 
tional and electronic properties ; in all applications of weighing (prima- 
rily determinations of atomic weight and of solubility^, etc.) differences 
in weight of the atoms are directly of use, and diffusion in the vapour- 
state also depends noticeably on the mass and even permits separation ; 
Aston* has thus succeeded in fractionating metaneon and neon. Centri- 
fuging also is a process in which mass plays a part and can be applied in 

1 H. Mo-SELEY, Phil. Mag. 26, 1024 (19i:}) : Ihid. 27, 705 (1914). Sec also A. 
VAN DEN Broek, Fluj-'i. Z. 14, 32 (1913). 

2 E. Rutherford and C. Andrade, riiil. Mag. .Ma\ (1914). 
^ K. Fajans, Naturwissenschaften 2, 544 (1914). 

* Aston, B)-iti-sh Association Report, Birmingham (1913). 



78 ADVEXTUEES IN RADIOISOTOPE RESEARCH 

several cases for separation. On the other hand, the theory mentioned 
above considers the chemical properties as essentially independent of 
the mass, and this applies also to the spectrum and radius of the atom. 
Differentiation between gravitational and electronic properties is 
naturally only clear-cut in limiting cases ; for example, the velocity 
of diffusion in liquids, which is primarily governed by the radius, is 
not independent of the mass^ and, according to Bohr, the same 
should also apply in respect of the Rydberg constant of the spectrum 
series^ ; a difference in atomic weight of 1 per cent affects the latter 
quantity by about 0.05 per cent. The characteristic vibrations of the 
molecules in the space lattice, and consequently the specific heats, also 
are probably noticeably different in isotopes^. 



2. CAN ISOTOPIC ELEMENTS REPLACE EACH OTHER CHEMICALLY? 

From the above discussions it is evident that isotopic elements are 
certainly not truly identical ; the question now is whether they can be 
denoted as chemically identical, i. e. whether they can replace each other 
in their chemical mass action. It is well known that the concentration 
of substances taking part in all chemical reactions is important (law of 
mass action of Guldberg and Waage) ; if isotopes are chemically 
identical the concentration must be represented by the sum of the 
isotopic elements present. For example, the solubility product of ba- 
rium-free radium-mesothorium chloride would be written in the form 

[Ra** -f mesothorium**] [Cl]2 = K 

Now there is a particularly clear method of testing for replaceability. 
In electrochemical processes a jump in potential is determined by the 
concentration of ions of the metal involved ; now when two elements 
(A and B) are replaceable, the addition of ions of the element B to those 
of A should exercise the same effect on the potential jump as if the ele- 
ment A had been raised to the ionic concentration A + B. For example, 
the potential difference RaD metal/RaD nitrate solution should be 
changed to the same extent by the addition of lead nitrate solution, 
within the meaning of Nernst's theory of the galvanic production of 
current, as if the ionic concentration of RaD had been increased, and 
vice versa. 

Instead of the electrode potential of a metal, the so-called decomposi- 
tion potential, which, according to Le Blanc, is of the same magnitude 



iG. Hevesy, Phys. Z. 14, 1209 (1913). 

2N. Bohr, Phil. Mag. 27, 512 (1914). 

3 K. Fajans, Naturwissenschaften 2, 544 (1914). 



THE PKOBLEM OF TlIK ISOTOPIC ELEMENTS 71)1 

and is the voltage at which the element can be deposited elect lolytically, 
can be considered. This was the first method which we adopted to solve 
the above problem, namely, to determine whether the decomposition 
potential of an element is displaced when an isotopic element is added 
to it. The sensitivity of radioactive methods permits the quantitative 
determination of even the unweighable amounts which always deposit 
l)elow the decomposition potential, and this opened up a second method 
of testing the problem ; we studied the variation in these amounts on 
adding isotopic elements. The third method depended directly on mea- 
suring the potential difference shown by a RaD peroxide electrode. 
More details will be discussed below concerning the method of depositing 
RaD peroxide which we have succeeded in preparing from radium ema- 
nation in visible amounts. 



3. STUDIES ON THE REPLACEABILITY OF ISOTOPES 

(a) The Decomposition Potential of Radioelements 

When determining the curve of the decomposition potential it is usual 
to measure the current passed by the cell as a function of the electrode 
potential. In plotting these curves it is always postulated that the 
current is carried essentially by the ion whose decomposition potential 
is to be determined and that current can pass continuously only when 
the potential difference attained at the cathode is equal to that which 
would be registered when the metal in question is immersed in the so- 
lution. This method of determining decomposition potentials is not 
applicable in radio-electrochemistry since the concentration of the radio- 
ions is not sufficient to carry the current exclusively. Therefore, we 
studied the amounts of the radioelements deposited during a time of 
24 hr, under precisely the same conditions, as a function of the cathode 
potential. In the first method a sudden increase in the current strength 
occurred at the value of the decomposition potential ; in the second 
method there was a sudden increase in the amount deposited ; a further 
difference between the two types of decomposition-potential curves 
consists in that the deposition in the second type can be investigated at 
potentials even higher than the decomposition potential whereas in 
the the first type the cathode potential does not rise even when the 
current is increased. 

We have plotted in Fig. I a decomposition-potential curve of 
the second type for radium-E ; the solution was about 10"^N in RaE 
(isotopic with bismuth). 

It is evident from the curve that some RaE is deposited at any potential 
and that because of the sensitivity of the method this amount can be 



80 



ADVENTURES IX RADIOISOTOPE RESEARCH 



determined quantitatively but that at —0.24 V (compared with the 
calomel electrode) there occurs a sudden increase in the amount de- 
posited. If bismuth nitrate is now added to the solution until the 
Bi + RaE normality of 10"^ is reached the characteristic increase takes 
place at —0.14 V i.e., about 100 mV lower (see Fig. 2). According to 
Nernst's theory it can be expected that a change in concentration of 



90 
80 
70 
60 

so 

40 
30 
20 



10 



*0-6 +05 *0-^ 



*6-3 *o-2 *o-i "-^^y *tT -k 'O-^ -OS -o-eyoff- 



Fig. 1. Cathodie deposition of radium-E. Concentration of the 
solution about 10"-»N in I^aE 



iOOir- 

90- 
80 
70 
60 
JO 
40- 
30 
20 
10 




*06 *0S *0^ *0-3 *0-2 *0-1 



-01 -02 -0-3 -04 -OS -(HVoit 



Fig. 2. Cathodie deposition of radium-E. Concentration of the 
solution 10" *N in bismuth isotopes, Bi -f RaE. 



THE PROBLEM OK THK ISOTOI'IC ELEMENTS 



81 



Irivalent Bi by a power often will result in a lowering of the decom- 
position potential by about 18 mV ; in the present example, therefore. 
9(1 mV would be expected. The break in the euive for pure RaE is in- 
de(Hl distinct but after all not so sharp as that for RaK -(- Bi ; this is 
an effect which in the first case is connected with Ihr lad 1 hat the elect- 
rode could not be covered with a layer of RalO (^xcn il' all Uic IvaK 
])i('S('nt were deposited. 



looir 




■K>6 ^-7 *0d *0-9 +1-0 +1-1 *f2 +1-3 *1A- +f5 *1-6 I/oft 



Fig. 3. Anodic deposition of Ihoiium-B peroxide. Concentialion 
of the solution IQ-i^N in ThB. 

The lack of sharpness becomes still more pronounced with more dilute 
solutions, e. g. in the case of our experiments with ThB. The solution 
was about 10~^'^N in ThB. The discontinuity for peroxide deposition, 
which can be traced more easily than that for metallic thorium-B, occurs 
at -|- 1.13 V (see Fig. 3). Since the decomposition potential in 0.001 N 
lead nitrate solution saturated with PbOg occurs at 0.87 V, the displace- 
ment amounts to 0.26 V. From the concentration difference of nine 
j)owers often a difference of 9 x 28 = 252 mV would l)e expected from 
theory, and thus the values agree very well^. Individual difficulties 
which have been encountered in these determinalions will !)(> examined 
in the discussion of the experimental details. 

1 If the average value of 20 mV determined by Gumming and Abegg {Z. Elektro- 
chein. 13, 19 [1907]) is assumed as the displacement per power of ten, then the 
agreement is less good, yet always passable in view of the largo sources of error 
in these experiments ; a similar mean value is obtained from our mcasuicments 
A\iiich are quoted later. 



b npTesv 



§2 ADVENTURES IN RADIOTSOTOPE RESEARCH 

(b) Deposition below the Decomposition Potential 

As was shown some time ago^, a small quantity of any radioelement 
deposits even below the decomposition potential and can be measured 
with the aid of sensitive methods which are now available. Thus, for 
example, 4 parts per 1000 of RaE are deposited at about —0.17 V in 
24 hr on an electrode 1 cm^ in area when the stirring is thorough ; this 
deposition is not affected by the presence of foreign ions, apart from 
bismuth, in the solution. If the solution is made 0.01 N in Bi ions the 
deposition of RaE no longer takes place. At a higher concentration 
the percentage deposition should naturally be much smaller ; 4 parts 
per 1000 of a 0.01 N bismuth nitrate solution would indeed amount to a 
few milligrams and thus would form a visible cover which cannot exist 
below the decomposition potential. This specific effect of bismuth ions 
on the deposition of RaE ions cannot be interpreted in any way other 
than by replaceability of these isotopes. 

We found similar results for ThB, irrespective of whether it was de- 
posited as metal or peroxide"^. For example, at 1 V i.e. below the deposi- 
tion potential, 5 per cent deposited and the deposition was in no way 
affected by the presence of thahium or other ions near to lead. In 10^^ N 
lead nitrate solution, the deposit was already less than I/2 P^rt per 1000 
and in 10"^ N the fraction deposited was no longer detectable. Clearly 
in this instance also, increased deposition occurs because of the high 
concentration, but the positions of most of the ThB atoms are taken 
by lead atoms, depending upon the concentration ratio of the two. 

(c) Measurement of a RaD Peroxide Cell 

Concerning the question of isotopy of the elements we are mostly limited 
to indirect methods Uke those described above, since no single instance 
is known in which both of two isotopes exist pure and in visible amounts. 
Visible amounts can be made available only from relatively long-Hved 
elements ; when recovered from minerals they are always contaminated 
with isotopes, e.g. uranium-II with uranium-I, ionium with thorium, 
mesothorium with radium, and so on. Radium-D, which occupies a posi- 
tion midway between the long- and short-lived, is always mixed with 
about ten million times the amount of lead when obtained from pitch- 
blende ; the considerable quantity of radium emanation available to 
us, however, gave us the opportunity to ol)tain directly visible amounts 
of RaD, completely free from lead because of its formation from 
emanation allowed to disintegrate in carefully purified quartz vessels. 

iG. Hevesy, Phil. Mag. 23, (528 (1912), 

2 The anodic doposition of ThB at strongly positive potentials was explained 
by the formation of (ThB)02 (F. Paneth and G. Hevesy, Si/?. Ber. Akad. Wi^-.s. 
Wien 122, 1027 [19131). 



TUE I'HOBLEM OF THE ISOTOPIC ELEMENTS 



83 



In the course of a few weeks the sealed flasks, which had meanwhile 
become coloured a (\cv]) brownish-violet, were opened, washed oul with 
nitric acid which had been distilled through a quartz condenser, and 
the solution was evaporated. Until completion of the electrolysis care 
was taken to use only quartz and no glass vessels. According to the con- 
ditions of electrolysis metallic RaD or RaD peroxide was obtained as 
a visible coating on small platinum wires ; ])reliminary experiments 
allowed this result to be expected since we had convinced ourselves that 
amounts of lead smaller than 0.001 mgm, as peroxide, are still clearly 
visible and electromotively effective, i.e. they can be used for building 
a cell^. We have manipulated various quantities of emanation, I/2 c 
on the avarage, but even 100 — 200 mc are sufficient for carrying out 
an experiment . 

The activity of the wires, as checked by measuring the a- and ^-radia- 
tion, was of the order of magnitude expected for pure lead-free RaD ; 
moreover, our apparatus was free from lead to the extent that we were 
able to detect an artificial contamination of 10-^ gm Pb. 

We measured the electromotive force of the following cell : 



Pt/RaD02/Ra(DN03)2, HNO3, RaDOo/KNOg/KCl, Bg^i^, H 
10-5N 10-3N satd. IN IN satd. 



The potential of RaD Og was found to amount to —0.884 V. The PbO, 
potential measured in the same conditions was found to be — 0.888 on 
the average^. 

In another series of experiments lead nitrate was added gradually 
and the following electromotive forces were found (at 20° C") (Table 1). 

Table 1 



Total noi-iiiality 

of the lead 

isotojies 


PbOj (Rad)Oj 


fHg 
(V) 


Change in 
potencial 
difference 


eSg 
(V) 


Change in 
potential 
difference 


10-s 

10-3 

10-1 


0.906 
0.774 
0.837 


0.032 
0.037 


0.906 

0.868 
0.839 


0.038 
0.030 




Total change 


0.069 


Total change 


0.068 



1 Refer to J. Koenig.sbebger and W. J. Muller, Phys. Z. 6, 849 (1905) ; 
Jbid. 12, COG (1911). 

2 The RaD nitrate conec^ntration (!Oul(l only Vjc determined to the nearest 
order of magnitude and therefore import anee should be attached only to tho 
agreement of the two potentials and not to their absolute values. 



e^* 



84 ADVEXTUKES IN RADIOISOTOPE EESEAKCH 

It is evident that the cells are identical within the limits of experi- 
mental error. We attach less importance to this than to the fact that 
the addition of Pb ions to RaD nitrate solution exercises precisely the 
same effect on the potential difference of the RaD peroxide which, 
according to Nernst's theory, the Ra D ions (and only they) should have. 



RT , c 

in 



is to be 



This proves that c in the Nernst formula 

^ nF C 

understood as the sum of the concentrations of the isotopic ions present. 
A special peculiarity of this RaD peroxide electrode deserves to be 
mentioned. If it is allowed to remain in contact with air for some time 
it immediately shows, on immersion, a potential which may be one- 
or two-tenths of a volt higher than the constant electrode potential 
established after a certain time. This is probably connected with the 
strong ionization in the vicinity of the wire. 

(d) Experimental Details 

The curves described above for the decomposition potential of RaE 
were obtained as follows : Two gold electrodes, each 1 cm^ in area, were 
immersed in 25 cm^ of O.IN nitric acid solution and were polarized for 
a long time until the desired electrode potential had established a con- 
stant value. A steady motion of the solution was ensured by passing a 
current of nitrogen. After the attainment of constant potential a few- 
tenths of a cubic centimetre of a solution at the same nitric acid con- 
centration and containing radium-E or RaE and Bi was added^ and the 
experiment was allowed to run for 24 hr. After this time the electrodes 
were withdrawn without interrupting the current, w^ashed with distilled 
water, always in the same way, and measured in an electroscope ; 5 cm^ 
of the solution were evaporated on a watch glass and likewise measured 
and hence the percentage deposition of RaE could be calculated. The 
experiments with ThB also were carried out similarly ; in this case the 
active solution was added to 100 cm^ of 0.001 N nitric acid and the de- 
position was made on correspondingly pre-treated platinum electrodes 
with an area of 4 x 2 cm^. The potential difference was measured hy 
means of a Siemens compensating apparatus. 

The RaE solution was obtained directly from emanation, and the 
thorium-B by exposure of a platinum foil to radiothorium. Particular 
care was used in the latter case to exclude lead completely ; a part of 
the experiment was carried out in quartz vessels anrl with the use of 
water purified specifically for this purpose-. 

1 The addition of small amounts of bismut h to make the solution about lO'^'X 
often caused an initial change of the cathode potential by several millixolts. 

2 The purification of the water was that usually employed for dctei mirations 
of atomic weicht (of. O. Honigschmid, Mitt. r/. Tn-st. Bndiitiujoy.schinnj. 8, S). 



THE PKOBLK.M OV THE ISOTOl'lC ELEMENTS 



So 



The ell'ect ol' adding very sniull amounts o! lead on the de})osition of 
ThB below its deeomposition potential was studied as well. 

Table 2 elearly shows the decrease in percentage deposition ol TliB 
from a O.OOIN nitric acid solution on platinum electrodes ( + 0.4 V, 
^Hg) ^vith increasing concentration of lead ; in every experiment four 
electrode surfaces were measured and the mean value was taken. 



Table 


2 






.Vmouia ol ThB 


■J'otal coiicoiit i"ati(jii of 
Pb isotojios 




deposited, as a 

percentage of that 

originally present 

(%) 


5 X 10-12 N. 




0.98 


10- » 




0.75 


10-' 




0.86 


10-5 




0.105 


10-3 


no longer detectable 



Thus up to a concentration of the solution of !()-' N, the deposition 
is only slightly affected, at IQ-^N a marked fall is already noticeable, 
and at IQ-^N the deposition is no longer measurable. This method, 
which can be still further refined by choosing smaller electrodes, still 
permits the detection of very small amounts of inactive lead, since the 
addition of another element, e.g. thallium, which is a neighbour of 
lead, has no noticeable effect on the deposition of ThB even at a con- 
centration of 10-3NT1. 



100%{- 
90 
80 
70 
60 
SO 
40- 
30- 
20 
10 



J_ 



/^ 



*0-6 *07 *0-8 *0-9 *1-0 *1-1 *1-2 *1-3 *H 



-TT^^TT^ 



■e/o/t 



Fig. 4. Anodic procipitulion oi TliBUg. The lead isotope concent- 
ration [Pb + ThB] of the solution lO-^X. 



86 ADVENTURES IN RADIOISOTOPE RESEARCH 

The determination of the decomposition potential by means of the 
methods mentioned above is based on the assumption that the current 
strength is large enough to permit deposition of the whole quantity 
of the radioelement within the duration of the experiment. It is easily 
seen, e. g. in the electrolysis of a 0.001 N lead nitrate solution, that the 
above condition is far from being satisfied, since the electrode potential 
is attained in our apparatus at a current strength of about 3 x 10"^ A 
which, in the course of 24 hr, is capable of depositing only a very minute 
fraction of the lead ions present. This is particularly emphasized since, 
if this point is not taken into consideration, there will be found too 
high a value in determining the decomposition potential by the methods 
mentioned (sudden increase in the amount deposited). For example, 
Fig. 4 shows the apparent decomposition potential of ThB using 0.001 N 
solution ; it is considerably higher than the calculated value, and the 
explanation is probably to be found in the reason mentioned above. 

We hope to be able to revert to several of the points which have been 
discussed, particularly to the deposition below the decomposition 
potential. 

4. DISCUSSION 

It has already been mentioned above that the difference in the atomic 
weights of individual isotopic elements exists without any doubt. Hence 
it follows that, in so far as gravitational properties are concerned, the 
isotopes are not identical and that by centrifuging, for example, meso- 
thorium should be easier to separate than its isotope radium from 
barium. On the other hand, a similar differential in the chemical pro- 
perties of isotopic elements is not observed, we have found replace- 
ability in the electrochemical behaviour. It is concluded that the electrode 
potential may be written in the form : 

_ RT Ec 

— ~z, In 

where Zr denotes the total concentration of all the isotopes present, 
and correS])()ndingly the mass action law may be written in th(> form : 

[Zisotope A]"' [i:isotope B]"^ ... 

— = A 



[Zisotope A']"' [Zisotope B']"^ . . 

The proposition that two atoms with different weights can replace 
each other in their mass action seems at first glance to contradict the 
second law of thermodynamics. The contradiction disappears, however, 
when the concept of chemical individually, to which the mutual replace- 
ability is related, is considered more closely and is defined appropriately. 



THE PROBLEM OF THE ISOTOPIC ELEMENTS 87 

We generally ascribe to each element a particular chemical character 
which varies discontinuously from one element to another. In their mass 
action silver atoms can replace only other silver atoms and not lead, 
thallium or other atoms. Accordingly, in the Nernst formula for the 
electrode potential of silver : 

RT , c 

f = In — 

nF C 

e can be changed only by the addition of silver ions and not by others. 
If it is now found that atoms which, in spite of having different atomic 
w<Mght, replace each other chemically and that c must be understood 
as the total concentration of the isotopes, it then seems necessary to 
define the concept of chemical individuality such that this does not 
imply complete equality of the atoms involved but the mutual replace- 
ability of the two atoms. The correlative of replaceability seems to be 
equality of the nuclear charge numbers whose fundamental importance 
becomes more and more prominent. 

Summary 

Experiments have been made to discover whether isotopic elements can replace 
each other chemically ; the following electrochemical methods have been emplojcfl 
for this purpose. 

(1) The electrolytic deposition of radium-E with and without the addition 
of bismuth has been studied and it has been found that the decomposition potential 
is displaced by the addition of bismuth in the sense and by the amount which 
Avould be expected of the addition of the same (RaE) ions in accordance witli 
Nernst's theory ; a study of the deposition of thorium-B with and without the 
addition of lead yielded the same result. 

(2) It has been shown that the deposition of the very small amounts of ladio- 
elements which precipitate below the decomposition potential is hindered by 
the presence of isotopes (and only by these), and this likewise can be explained 
only by replaceability. 

(3) Radium emanation has been allowed to disintegrate in quartz and the 
radium-D formed has been deposited electrolytically as the peroxide on platinum 
wires ; visible and at the same time electromotively active amounts (a few- 
thousandths of a milUgram) have thus been prepared. The cell RaDOg I RaD(N03)2 I 

I KNO3 I KCl,Hg2Cl2,Hg showed the same electromotive force as a cell similarly- 
made with lead peroxide, and furthermore the addition of lead ions to the RaD 
solution changed this e. m. f. in the same way a corresponding addition of 
RaD ions should change it according to Nernst's theory ; hence it is conducted 
that the ionic concentration c in tlie Nernst formula 

RT , c 
nF C 

must be understood as the sum of the isotopic ions. 

From Our study, therefore, the conclusion must be diawn that isotopic elements 
are able to replace each other in their mass action. 



88 ADVENTURES IN RADIOISOTOPE RESEARCH 



Comment ok paper 6 

As shown by Nernst the electrode potential of a metal is proportional to the 
logarithm of the concentration of its ions present in the suri'ounding solution. 
The validity of this regularity was tested up to 0.0001 N ionic concentration. 
The application of labelled bismuth and labelled lead permitted us to demonstrate 
the validity of this regularity at much lower ionic concentrations than the above 
mentioned one. When this investigation was carried out in 1913 the notion of 
isotopes had just emerged, and it was thus of interest to demonstrate that the 
voltage at which RaE, for example, is precipitated on the cathode, is influenced 
by the addition of a bismuth salt to an extent to be expected on assuming the 
practical chemical identity of bismuth and RaE. It is not, however, influenced 
by adding salts of other metals. Below its decomposition voltage minute traces 
of RaE aie deposited as well, this minute precipitation is also influenced by 
addition of bismuth salts, but not by addition of salts of other metals. 

The large amount of radon available at the Vienna Institute made it possible 
lo obtain a visible RaD layer on a platinum wire. The electrode potential of a 
RtiDOj electrode was found, measured against a calomel electrode undistinguishable 
from the potential of a lead peroxyde electrode. The experiments were carried 
out with peroxide of lead instead of metallic lead, as the electrode potential of 
metallic lead was found not to be sufficiently reproduceable. 

If it weie possible to measure these electrode potentials to an accuracy of 
several decimals, we would piesumably measure some diffeience between the 
electrode potential of lead peroxide and ladium D peroxide, as isotopes are not 
strictly identical in their chemical properties. The very far-reaching practical 
chemical identity of isotopes of an element is, however, conspicuously demon- 
strated by the lesults of this paper. 



Originally jnihlislKMl in Z. phtj.s. Chciii. 89, 294 (1914) 

7. THE VELOCITY OF DISSOLUTION OF MOLECULAR 

LAYERS 

O. TIevesv and E. Rona 
From the Clieniical Institvite of the Univeisity oi" Budapest 

The vf^ocity of dissolution ol' finite layers can be followed quantitatively 
by considering the process of dissolution as being comprised of two 
partial processes ; one of these consists in the formation of a layer ol" 
saturated solution surrounding the solid surface and the other is a 
process of diffusion from this boundary layer into the liquid^. 
The velocity of dissolution is represented by the equation : 

dx/dt = DOF{r^ - c)ld 

where d denotes the thickness of the boundary layer, F a proportionality 
factor, D the diffusion coefficient, the area, c^ the saturation con- 
centration, and c the concentration of the solution. 

The dissolution will therefore proceed more rapidly the smaller Ihe 
thickness of the boundary layer, i.e. the greater the speed of stirring, 
the greater the diffusion velocity of the participating molecules and the 
I'urther the solution is from the saturated state ; the formula also shows 
a parallelism between solubility and velocity of dissolution. 

Nernst and Brunner^ have shown that these ideas are quite generally 
applicable to heterogeneous reactions. 

The present communication discusses the course oi Ihe dissolution 
process of molecular layers, which could be also described as infinitely 
thin, and the extent to which the above simple equation is satisfied. 



THE PREPARATION OF INFINITELY THIN LAYERS 

It is well known that an infinitel}^ thin layer of radioactive metal or 
its oxide, known as the so-called active deposit, can be obtained simply ; 
by the decay of the gaseous emanations metallic products are formed, 
which are isotopes of polonium, lead, ])ismuth and thallium and which 



1 NoYEs and Whitney, Z. phy.s. ('hem. 23, (i!S9 (1897). 

2 Nernst and Brunner, Z. phy.s. Chan. 47, o2, oO (1904). 



90 ADVENTURES IN RADIOISOTOPE RESEARCH 

gradually become deposited from the suspension in the air ; this de- 
position process can be considerably accelerated by applying an electric 
field. In our experiments we made use of a radiothorium preparation, 
which provides a constant source, and the active deposit yielded by the 
emanation was collected on a quartz surface 1.6 cm in diameter. The 
quartz disk was covered with a mixture of ThB (lead isotope) and ThC 
(l)ismuth isotope) because the first decay product of emanation, ThA, 
decays very quickly with a half-life of ^1^ sec. A simple calculation yields 
5 X 10-11 gm as the total mass of the deposit, of which about 90 per cent 
consists of ThB and 10 per cent of ThC. In order to cover the surface 
completely with a molecular layer of lead 2 x 10^^ gm would be necessary, 
i.e. 50,000 times the amount actually present ; we can thus rightly 
consider tiie surface as having an infinitely thin covering of lead and 
bismuth. 

The velocity of dissolution was determined as follows : The quartz 
disk was allowed to stand for several hours after cessation of the activation 
until radioactive equilibrium had been established and the /^-activity 
formed by the deposit, and showing the relative amounts of Pb and Bi 
present, was then determined ; the disk was then placed in a bell- 
shaped vessel, provided with an outlet tube and cock, and after a certain 
time the 100 cm^ of liquid in the vessel was drained out. Care was taken 
to attain a constant stirring speed, the disk being placed in the solution 
only when this speed had been established. 

The /3-activity of the quartz disk treated in this way was then measured 
again 15 min after completion of the experiment and at various later 
time intervals, and it was thus possible to decide upon the amounts of 
Bi and Pb present, from the change of the activity with time, and to 
determine the percentage which had entered into solution. 



THE DETERMINATION OF THE VELOCITY OF DISSOLUTION 

The investigation was concerned with the determination of the effect 

(1) of the concentration of acid in the solution 

(2) the viscosity 

(3) of the speed of stirring 

(4) of the time 

on the velocity of dissolution of the molecular layer and finally on the 
effect due to isotopes of the corresponding elements in the solution. 
The percentages of Bi and Pb isotopes which dissolve in nitric acid 
in 60 sec, under the same experimental conditions, are summarized in 
Table 1. 



THE VELOCITY OV DISSOLUTION OF MOLECULAIl LAYERS 



91 



Tablk 1. — A^vioTN'Ts Dissoi.vKn IX ()0 sec 





In 


10-' N 


10-' K 


10-»N 


10-» N 


10-' X 


X 




water 


HNO3 


HNO3 


HNO3 


HNO, 


nNo, 


HNO, 




(%) 


(%) 


(%) 


(.%) 


(%) 


(%) 


(%) 


Bismuth 
















isotope 


37 


38 


35 


61 


72 


77 


78 


(ThC) 
















Lead isotope 


60 


61 


60 


80 


81 


83 


84 


(ThB) 

















The velocity of dissolution is the same in 10~* N acid as in conductivity 
water but increases with further increase of the acid concentration and 
in N acid amounts to about twice the above value. It is well known 
that the bismuth isotopes dissolve colloidally in water and from diffusion 
experiments the conclusion was drawn that this is no longer the case 
in 10~3 N acid^. It suggests itself to associate the sudden increase in 
Ihe velocity of dissolution with this change. 

The velocity of dissolution of finite layers depends on, among other 
factors, the diffusion velocity of the products involved. In order to 
change this velocity glycerol was added to the nitric acid in order to 
cause a considerable increase of the viscosity and a corresponding lower- 
ing of the diffusibility of the hydrogen and other ions without otherwise 
changing the experimental conditions. The consequence of the glycerol 
addition was. as is clear from Table 2, a decrease in the velocity of 
dissolution. 

Table 2 



Dissolved in 
K)-' N HXO, 



Dissolved in 10— ^X 

HXOj containing 

25% glycerol 



Bismuth 






isotope (%) 


61 


52 


Lead 






isotope (%) 


80 


73 



The viscosity of the glycerol mixture found by the ordinary outflow 
method was 1.650 relative to that of water as unity. 

Even after treatment for several hours with concentrated acids it is 
found that the quartz disk still retains about 20 per cent of its original 
ThB— ThC coating which, however, is not present on the surface but is 
situated inside the quartz where it has arrived through the so-called 



^ F. PA>rETH, 

(1913). 



Kolloid-Z. 13, 1, 297 (1913); G. Hrvp:.sy, Phys. Z. 14, I2U9 



92 ADVENTURES IJT RADIOISOTOPE RESEARCH 

radioactive recoil process. A portion of the active deposit is laid down 
on the quartz disk in the form of ThA, the ThA then emits a-particles 
with the result, according to the principle of action and reaction, that 
a recoil of the atoms is required and thus some of the ThB atoms formed 
from ThA are deposited under the surface of the quartz. The range of 
such recoil atoms amounts to about ^/jq cm in air and, therefore, less 
than 10-* cm in quartz ; this thin layer of quartz is quite sufficient 
to protect the fraction driven inward by the recoil effect from the reaction 
of the acid, although radiation, from which its presence can be inferred, 
still affects the electroscope through this layer. 

The portion of the active deposit found underneath the surface 
depends on the time and other conditions of exposure Avhich have been 
chosen to be strictly the same in all these experiments. The portion of 
the active deposit occurring below the quartz surface, found experiment- 
ally to be 20 per cent, was not taken into account in compiling the tables, 
the values in w-hich refer only to the soluble part of the active deposit. 

The determination of the effect of the stirring speed on the velocity 
of dissolution meets with difficulties. Because of the large velocity of 
dissolution of molecular layers the times of experiment must be limited 
to a few minutes and the unavoidable immersion and withdrawal of the 
quartz disk from the solution always acts as intense stirring. In our 
experience the effect of stirring velocity on the velocity of solubility of 
molecular layers was not considerable. 

The Relation between Velocity of Dissolution and Solubility 

As shown by the above formula, the velocity of solubility of finite 
layers increases with the solubility of the substance ; this is true also 
for infinitely thin layers, and thus the velocity of solubility of the lead 
isotopes is greater than that of the bismuth isotopes (Table 1), both 
in water and in nitric acid, corresponding to the greater solubility of 
the lead salts involved. 

Lead peroxide dissolves more slowly than metallic lead and lead 
monoxide, in agreement with its lower solubility in HNO3. Such peroxide 
layers^ were produced by the anodic deposition of ThBOg on platinum. 
Only 20 per cent of ThBOg dissolved in the same conditions in which 
80 per cent ThB entered the solution. Since dissolution from quartz is 
not strictly comparable with dissolution from platinum, a comparison 
was therefore made between the velocity of solubility values of ThBOg and 
ThB likewise deposited on platinum by a cathodic reaction ; in this case 
also more of the latter dissolved as is proved by the figures in Table 3. 

IF. Paneth and G. Hevesy, Wien. Ber. 122, 1038 (1913). 
2 F. Paneth and G. Heatesy, Wien. Ber. 123, 1050 (1913). 



THE VKLOCITV OK DISSOLUTION OF MOLECI'LAR LAYERS 93 



Table 3 



llatios 


oi tlie amounts of TUB 


Ratios 


o£ 


tlie 


amounts of TliC 




and 


ThUO. dissolving in 


and 


ThCOo 


?) dissolving in 






1 mill 






1 


milt 


10-2N 




10-2N HNO3 


10-2N 






10-2N HNO3 


HNO3 




+ 10-iN 
oxalic acid 


HNO3 






+ 10- IN 
oxalic acid 


4.0 




0.76 


3.1 






0.75 



ll is also evident thai in the presence of a reducing agent sucii as 
oxalic acid the large difference between the dissolution velocities of the 
(•atliodically and anodically deposited lead isotopes disappears, in 
agreement with the ready solubility of the peroxide in oxalic acid solution. 

In the more recent development of electrochemistry particular atten- 
tion is devoted to the reactions wdiich take place between the deposited 
products and the electrode material ; the study of the velocity of 
solubility with the elect rolytically deposited radioelements offers an 
easy method of approaching more closely to these problems ; thus, in 
the case of polonium it was proved that this element forms stable com- 
])ounds more easily with Pt and Pd than with gold. 

Change in the Dissolution Velocity due to the Presence of Isotopic Ions 
of the Dissolving Metal in the Solution 

Isotopic atoms are interchangeable in their electrochemical reactions^ 
Now the dissolution of a metal is to some extent the reverse of its electro- 
lytic deposition and therefore it is to be expected thatThB will dissolve 
only to the same small extent as lead in nitric acid saturated with lead 
nil rate. 

Before discussing the behaviour of a molecular layer when dissolving 
in a solution which already contains some dissolved isotope, we w^ould 
like first to explain in more detail the process which takes place between 
a solid phase and its saturated solution. Just as the equilibrium state 
between a liquid and its saturated vapour is regarded as dynamic, i.e. 
the assumption is made that the same number of molecules condense 
from the vapour and leave the liquid in unit time, the equilibrium state 
between a solid phase and its saturated solution is also regarded as 
dynamic, i.e. it is assumed that at the boundary of, for example, 

PbC'lg solid I water saturated with PbC'lg 

a dynamic exchange of PbClg molecules takes place between the two 

iG. Hevesy hihI F. Paneth, Phy-s. Z. 15, 7!J7 (1914). 



94 ADVENTURES IX EADIOISOTOPE RESEARCH 

phases. It is necessary to know the rate of this exchange because if it is 
very large the (ThB)C'l2 molecules in the solid phase will exchange 
directly with tlie Ph( U molecules of the solution and thus simulate 
direct dissolution. 

An answer to this question would be possible if, for example, a 
saturated lead chloride solution could be shaken with solid PbClg, the 
lead atoms of which were numbered or characterized in any other way 
without affecting their chemical properties, by finding in which phase 
the numbered atoms then existed. vSuch ideally labelled lead atoms are 
the radioactive isotopes (ThB, RaB, AcB, RaD, etc.). We need only 
add, for example, {ThB)(N03)2 to the solution of lead nitrate, precipitate 
the Pb— ThB mixture as chloride and thus to obtain a "coloured" lead 
chloride. If 1 mgm of PbCIg were originally associated with one relative 
unit the detection of this relative unit in the saturated solution would 
allow the inference that 1 mgm of the lead atoms originally in the solid 
phase had then been transferred into the saturated solution, or vice versa. 

The application of radioactive indicators thus serves to permit tracing 
of the exchange of atoms of the same kind between two phases ; the 
"colouring" of the labelling atoms is a purel}^ radioactive property and, 
although their mass is different from that of the labelled isotope (they 
have different atomic weights), their chemical reactions, with which 
we are concerned, are still the same^. 

To determine the velocity of exchange between the solid lead chloride 
and its saturated solution, 250 mgm of ThB-labelled PbClg was shaken 
with 25 cm^ of a saturated solution of pure lead chloride in a thermostat 
at 20°C. After 24 hr the mean value of ten experiments showed that 
1.2 mgm, or ^2 V^^ ^^^^ of the lead chloride originally in the solid phase 
had entered the saturated solution ; after 48 hr the value was about 
'/4 per cent. The lead chloride solution was prepared by cooling an 
originally slightly supersaturated solution in the thermostat and was 
therefore fully saturated ; the possible sources of error all tended to 
yield high values for exchange and the above value should therefore 
be regarded as an upper limit only. 

The velocity of exchange depends very markedly on the mechanical 
consistency of the solid phase, and this also applies to the velocity of 
solubility-. It is thus imperative to compare these two quantities under 
the same experimental conditions. For example, on shaking 250 mgm 



1 These ideas do not apply strictly to diffusion processes or, therefore, to the 
exchange of atoms in the same phase, because the velocity of diffusion is dependent 
on the mass ; this dependence is very slight, however. Considered from the stand- 
point of diffusion only those atoms which are both isotopic with and of tlie same 
mass as those under study are really ideal indicators. It appears, however, that 
UY may be such an ideal indicator for UXj. 



THE VELOC'ITV OF DISSOLUTION OF MOLECULAK LAYERS 



95 



of the same lead chloricU^ 44 per cent had already dissolved in 1 hr, 
representing 44 per cent of the saturation concentration since the liquid 
volume amounted to 25 cm^ and 250 mgni are soluble in tliis volume. 

It is seen, therefore, that tlie velocity of exchange belween the 1 wo 
phases is small compared Avitli the velocity of solubility. Il will be seen 
later that in the case of a molec^ular layer the exchange velocity becomes 
much larger when expressed on a percentage basis but is still smaller 
than the velocity of solubility, and thus a diminution of the velocity 
of dissolution of a substance can still be detected by the presence of its 
isotope in the solution. 

To this end we have compared the amounts of ThB collected on 
quartz which dissolved in water and in a saturated PbClg solution, under 
the same conditions, in 1 hr. There w^as only a small difference since in 
the first case 79 per cent dissolved while in the second the value was 
75 per cent. 

Shorter experimental times were then chosen and thus the presence 
of lead ions in the solution had a very considerable effect on the velocity 
of solubility of ThB. These experiments were performed with the same 
apparatus used for obtaining the values recorded in Table 1. 



TaULI-; 4. — ExpERIMENT.JiI. TIME 60 SGC 



Bismuth isotope ThC 
Lead isotope ThB 



.Vmount ilissolved 

in 10 - 3 N HXO., 

(%) 



Amount dissolved in 

10—' N HXOj s:iturated 

with Pb(X03)2 

(%) 




fi4 
67 



(■-'; 



The presence of lead ions in the solution diminishes the velocity of 
solubility of the lead isotope but not that of the bismuth isotope. It is 
a fortunate circumstance that ThB and ThC are simultaneously present 
in the same place on the quartz surface, and when, in spite of this, the 
velocity of solubility of only one is affected, this means that there is 
a specific effect due to the addition of the appropriate element ; for 
example, glycerol, which has no selective effect but which increases the 
viscosity of the solution, affects the velocity of solubility (Table 1) of 
ThB and ThC equally. 

In order to ascertain whether a small concentration of lead ions 
affects the amounts of ThB and ThC dissolved, w^e have performed 
experiments in 10~3N HNO3 solutions which were also 10~^N in lead. 
The time of experiment was 40 sec and the arrangement was different 
from that described above. Th(> ratio of the amounts of l^b and Bi 
dissolved was found to be : 



96 ADVENTURES IN RADIOISOTOPE RESEARCH 

in piiro 10-3N HNO3 1.99 

in 10-3N Pb(N03)2 + IQ-^N HNO3 1.49 

which is still a significant difference. 

The conclusion to be drawn from these experiments is that the velocity 
of exchange between the solid phase and its saturated solution is already 
commensurate with the velocity of dissolution for a molecular layer, 
but, that when the experimental time is short an effect on the velocity 
of dissolution due to the presence of isotopic ions in the solution can 
l)e detected. 

The following experiment seemed to be of interest in connection w ith 
those described above: 200 mgm of Pb(N03)o labelled w^ith ThB was 
added to a solution of PbClg (200 mgm), a portion of the PbClg was then 
allowed to crystallize out and the distribution of the different kinds 
of lead atoms between the chloride and nitrate was studied. After a few 
minutes required for performing the manipulations it was shown that 
there was a completely uniform distribution of all the lead atoms, 
within the limit of error amounting to 1 per cent. 

Z. KlemeisSiewicz^ has recently performed similar experiments, 
lie studied the distribution of ThB and also RaB between a lead amal- 
gam and a mercuric nitrate solution and found a completely uniform 
distribution ; the accuracy of his experiments was greater, with an error 
of Yo per cent. 



Summary 

The velocity of dissolution of molecular (infinitely thin) lajers shows quali- 
tatively the same behaviour as that of finite layers. The velocity of dissolution 
of lead and bismuth isotopes in nitric acid increases with acid concentration, 
with lowering of the viscosity of the solution and with the sohibility of the 
suVjstance involved. 

The presence of lead ions in the solution lowers the velocity of dissolution 
of the lead isotope ThB without affecting that of the bismuth isotope ThC. 

The velocity of exchange between the molecules of solid lead chloride and a 
saturated lead chloride solution can be determined by labelling the lead chloride 
with ThB ; in the case of a finite layer it is vanishingly small compared with 
the velocity of dissolution but in the case of a molecular layer the two properties 
are commensurable . 



^Z. Klemensiewicz, C, B. Accd. ScL, Paris 158, 1889 (1914) 



Originally piiblishod in Phu-s. Z. 15, 797 (1915) 

8. THE EXCHANGE OF ATOMS BETWEEN SOLID 

AND LIQUID PHASES 

G. Hevesy 
From the Institute for Jiadium Research of the Academy of Sciences of Vienna 

When a liquid is in contact with its saturated vapour there will 
take place, in accordance with kinetic ideas, a constant exchange bet- 
ween the molecules in the two phases. Correspondingly, it is to be ex- 
|)ected that a kinetic exchange of the molecules will likewise occur when 
a solid phase is in contact with its saturated solution. 

Radiochemical methods permit an experimental study of this ex- 
change. For example, if the exchange between the molecules of a solid 
layer of lead chloride and a saturated solution of lead chloride is to be 
determined the following procedure is adopted : A known amount, in 
relative (electroscopic) units, of ThB is added to a solution of known 
Pb(N03)2 content and the whole is precipitated with hydrochloric acid. 
In accordance with all previous experience the ThB can no longer be 
removed chemically from such a mixture of PbClg and ThBClg ; if there 
is, for example, one atom of ThB mixed with lO^*^ lead atoms on the 
average in this mixture, this ratio will remain the same after any che- 
mical operation and if a ThB atom can be detected electroscopically in 
the lead chloride phase which was previously free from ThB the con- 
clusion can be drawn that 10^° of the lead atoms originally mixed with 
the ThB have also entered this phase. Thus the ThB or another isotope 
of lead serves as an "indicator" for lead. 

If solid lead chloride labelled with ThB is shaken with a saturated 
(unlabelled) solution of lead chloride for 36 hr at 20°C it is found 
that less than 14 pcr cent has been transferred from one phase into tlie 
other. 

The determination of the velocity of dissolution of lead chloride having 
the same grain size showed that 44 per cent of the amount of PbCU 
corresponding to saturation passed into solution within 1 hr ; since 
the number of exchanged molecules (expressed as a percentage of all 
those present) is extremely small it appears that the velocity of exchange 
of lead chloride molecules between solid lead chloride and the saturated 
solution of PbC'lg is vanishingly small compared with the velocity of 
dissolution of solid lead chloride. 

/ Ilovesy 



98 



ADVENTURES IN RADIOISOTOPE RESEARCH 



A different result is obtained, however, if a study is made of the ex- 
change, not between a finite layer of lead chloride and its saturated 
solution but between a molecular film of lead chloride, which can easily 
l)e prepared by a radiochemical method, and the saturated solution. 
It is then seen that the percentage of exchanged molecules is very con- 
siderable and the velocity of exchange becomes commensurable with 
the velocity of dissolution of the molecular layer. 

This result can be expected from kinetic considerations ; a rapid 
exchange can take place, in general, only in the superficial layers. 

A lead rod, 4 cm long and 5 mm in diameter, was immersed for 1 min 
in 10 cm^ of a lead nitrate solution labelled with a known amount of ThB, 
and then the quantity of ThB deposited on the lead surface was deter- 
mined. This gives the number of lead ions originally in the solution 
which have thus been transferred to the lead surface. The first column 
of Table 1 records the normality of the Pb(N03)2, the second the number 
of lead ions per thousand originally in the solution and then occurring 
on the surface of the lead, the third the amounts of lead in grammes, 
the fourth the amount, expressed as a fraction, of that required, accord- 
ing to MtJLLER and Koenigsberger^, to indicate the potential of lead 
peroxide. On the basis of the Loschmidt number the mass of a unimole- 
cular layer of PbOg is calculated^ as 3.2 x 10"'^ gm. According to the 
measurements of Koenigsbeeger and Mijller^ twice this mass is re- 
quired for optical detection and eight times to impart the PbOg poten- 
tial to an area 1 cm^ in extent. 

Table 1 



Normality of the 

solution in 

Pb(N03)» 


Promille of lead 

of the solution 

transferred to the 

solid phase 


Amount of lead 

exchanged 

(sm) 


Number of molecular layers 

1 cm" in area which can be 

covered by the amount of 

exchanged lead 


10-5 


4.3 


4.4 X 10-^ 


0.069 


10-" 


4.5 


4.6 X 10-' 


0.72 


10-3 


3.9 


4 X 10-6 


6.2 


10-2 


3.7 


3.8 X 10-^ 


59 


10-1 


1.7 


1.7 X 10-4 


266 


1 


0.4 


0.4 X 10-* 


625 



According to our ideas on the process of the galvanic production 
of current, lead will either go into solution or will be deposited, when a 
lead rod is immersed in a solution of lead nitrate, according as the con- 
centration of lead nitrate is on one or the other side of the limit at which 



1 cf . W. J. MuLLER and J. Koenigsberger, Phys. Z. 6, 849 (1905). 

2 J. Koenigsberger and W. J. Muller, Phys. Z. 12, 606 (1911). 



THE EXCHANGE OF ATOMS BETWEEN SOMD AND LIQIII) I'HASES 99 

a potential difference of zero prevails between the lead nitrate solution 
and the metallic lead (absolute zero point of the electrolytic potential). 
Thus it might be thought that the amount of lead deposited in the above 
experiments does not represent the exchange between the two phases 
but is the result of such an unbalanced electrolytic process. The calcu- 
lation of the amount which can be expected to be deposited at an isolated 
electrode shows, however, that the value is much smaller than that 
actually observed. 

The capacity of the douljle layer at 1h(^ metal-electrolyte boundary, 
from the measurements of Krijger and Krumreich^, amounts to 27 
/.iF ; the potential difference of this condenser in the case of Pb/Pb(N03)2 
is always less than -^ 0.2 V ; thus the calculated charge of the condenser 
is 5.4 X 10~^ C. This quantity of electricity corresponds to 5.6 x 10-^ gm 
Pb , which is considerably less than the deposit of lead found by experiment . 

In order also to confirm experimentally that an exchange of atoms 
between the two phases, and not a one-sided deposition, is involved, the 
following experiments were performed : 

A lead rod similar to those used in the experiments already mentioned 
was coated electrolytically with a layer of metallic lead labelled with 
ThB, immersed in 10 cm^ of lead nitrate solution for 1 min and then, 
by determining the ThB content of the solution, the number of lead 
atoms transferred from the metallic phase into the lead nitrate solution 
was determined. 

Thus it was possible to establish that while 1.7 X 10^^ gj^-^ j^^j depos- 
ited from 10 cm^ of a 0.1 M lead nitrate solution on an area of 2 cm^ 
in 1 min., 1.6 X 10"* g Pb had correspondingly entered the solution in 
identical conditions in the experiment just mentioned. 

In our experiments, therefore, there is indeed an exchange of atoms 
between the metallic phase and the lead nitrate solution. Because of 
the magnitude of the amounts exchanged, which in certain conditions 
amount to one hundred times the unimolecular layer, the process cannot 
be a pure "kinetic" exchange (exchange at complete thermodynamic 
equilibrium) but involves nonuniformity of the lead surface and pre- 
cipitation of the lead atoms at particular points from the solution. 
The observed exchange is essentially a result of "local currents". 

The velocity with which the exchange of the lead atoms takes 
place in the interior of the solid metallic phase can be computed approx- 
imately since it is equal to the velocity of diffusion of lead in solid lead. 

The diffusion velocity of lead in mercury according to M. v. Wogau^ 
amounts to 0.6 cm^ hr"i at 18°C ; in solid lead the value is many times 



1 Kruger and Krumreich, Z. Elektrochem. 19, 020 (1913). 

2 M. V. WoGAr, Ann. Pluj-s. 23, 34."", (IflOT). 



100 



ADVENTURES IN RADIOISOTOPE RESEARCH 



less since the viscosity of solid lead is very much greater than that of 
liquid mercury^. The viscosity of mercury is 0.016 at 18°C whereas the 
value for solid lead, according to Kurnakow and Zbmaczny^, is 3 x lO^-; 
the velocity of diffusion of lead in solid lead calculated from these 
figures is 2 X 10~^^ cm^ hr-^^. The minuteness of this diffusion velocity is 
best brought out by means of the following analysis : Consider a diffu- 
sion cylinder consisting of four equal parts, each part being surrounded 
by four molecular layers with an assumed thickness of 0.8 x 10~' cm 
and with the lead atoms of the lowest four molecular layers labelled. 
After 1 hr there will be less than one per thousand of the labelled lead 
atoms in the uppermost part of the diffusion cylinder, in the third part 
only 1.6 per cent. The kinetic exchange during 1 min can extend only 
to the uppermost molecular layer and to a small extent to the second 
and third layers. 

Table 2 records the amounts of lead which have exchanged bet- 
ween a 0.001 N Pb(N03)2 solution and a lead rod in various times. 

Table 2 



Time 
(sec) 



Amount of lead 

exchanged 

(am) 



Number of molecular 

layers 1 cm^ in area 

which the exohan(?ed 

lead can cover 





15 


2.0 X 10- « 


3.1 




15 


2.3 X 10-6 


3.3 




30 


2.8 X 10-« 


4.4 




30 


2.8 X 10-« 


4.4 




60 


3.5 X 10-« 


5.5 




60 


3.6 X 10- « 


5.6 



Table 3 contains the results of such experiments in which a lead rod 
dipped into 10 cm^ of a 0.1 N solution of Pb(N0.j)2. 



Table 3 



Time 


.Vmount of lead 

exchanged 

(gm) 


Number of molecular 
layers 1 cm= in area which 


Ymin") 


the exchanged lead can 
cover 


1 


1 X 10-* 


156 


10 


2.1 X 10-* 


328 


30 


3.2 X 10-* 


564 



•■'How fur such an oxtrapolalion is permissible for the sohd state will slioitly 
be discussed on the basis of experiments. 

* Kurnakow and Zemaczny, Jh. Radioakt. 11, 25 (1914). 



THE EXCHANGE OF ATOMS JJETWEEN SOLID AND LIQllD PHASES 



101 



Difi'eiont behaviour is Ibund in studying lead peroxide suiiaces 
immersed in a lead solution labelled witliTliB. In this ease the exehange 
is much less ; between a PbOg surface 2 cm- in area and 10 cm^ of a 
0.001 N Pb(N03).^ solution, containing 0.001 N IINOg and saturated 
with similarly lalx^llcd PbOa. the following exchange takes place : 



Table 4 



Time 


Aiiiount o£ lead 

exchanged 

(gm) 


Expressed as 

molecular layers 

1 cm* in area 


Jixpressed in fractions of 
the amount required to 
impart the TbO., poten- 
tial to the area of 1 cm- 


10 sec 
1 mill 
10 mill 
60 min 


1.6 X 10-7 
2.4 X 10-' 
1.0 X io-« 
2.0 X 10-« 


V4 
V3 

1.5 
3.1 


1/ 
,'32 

/24 

V, 



Table 5. — The time of experiment in this ca.se is always 1 min ; the concentra- 
tion OF the labelled lead nitrate solution varies between 10-1 _^jji5 10-6 X 



Normality of the 

solution of lead 

nitrate 


1 
Amount of lead j Expressed in 
exchanged 1 molecular layers 
(gm) j 1 cm^ in area 

i 


Ex-pressed iu fractions of 
the amount required to 
impart the PbOj poten- 
tial to the area of 1 cm 


10-6 
10-5 

10-3 

10-1 


0.64 X 10-* 
4.0 X 10-* 
3.0 X 10-« 
2.2 X 10-' 


Vio 
3.5 


/go 

V48 
V'l6 
V2 



Here also an exchange rather than a unilateral dissolution is involved 
as is proved by the following experiments : This time a labelled Pb02 
surface 2 cm^ in area is immersed in 10 cm^ of a 0.001 N Pb(N03)2 solu- 
tion, saturated with PbOa and containing 0.001 N HNO3, and it is found 
that the following amounts of lead (Ta])le 6) have passed into solution 
from the solid phase : 

Table 6 



Time 



Amount of lead 
exchanged 
(gm) 



Expressed in 

molecular layers 

1 em" in area 



Expressed in fractions of 
the amount required to 
impart the PbOj poten- 
tial to the area of 1 cm' 



10 sec 


1.5 X 10-' 


v* 


1 min 


2.8 X 10-' 


V3 1 


20 min 


O.S X 10-^ 


1.2 ! 



32 
24 
6 



The experiments just described are made difficult owing to the break- 
ing off of invisible amounts of lead peroxide which fall into the solution 



102 ADVEXTUKES IX EADIOISOTOPE RESEARCH 

and are co-determined when the solution is evaporated, thus producing 
an erroneously high exchange. 

In the experiments discussed here it was merely assumed that lead 
and ThB cannot be separated by chemical and electrochemical reactions, 
as was first proved by Fleck and later confirmed by many authors. 
If one phase contains on the average lO^o atoms per atom of ThB and 
if we can detect a ThB atom in the other phase which was originally 
free from ThB then, as already mentioned in the introduction, the con- 
clusion can be drawn that lO^o lead atoms also have been transferred 
from the first to the second phase. Our experiments do not indicate how 
many atoms have changed places more than once between the two phases. 

It should be mentioned that when diffusion processes are involved the 
presence of one ThB atom cannot strictly be taken to imply the accom- 
paniment by 10^0 atoms of lead, since the diffusion velocities of ThB and 
Pb are not equal. As far as solid and liquid phases are concerned, however, 
in which the diffusion velocity is very little dependent on the mass, it 
is practicable to draw the above-mentioned conclusion and, for example, 
to equate the velocity of diffusion of lead isotopes in lead to that of lead 
in lead. 



Summary 

The exchange of atoms between two phases, for example, between metalhc 
lead and a lead nitrate solution, can be followed by labelling the lead in one 
phase with one of its isotopes, for example, with ThB; the amount of labelled 
lead transferred in a given time into the other phase can then be determined. 

In the case of Pb/Pb(N03)2 the exchange is very rapid and depends mainly 
on the local currents. At particular points in the metal some lead goes into solution 
and at other places lead is deposited from the solution. 

The exchange between a surface of lead peroxide and a lead nitrate solution 
is much less ; in the experimental conditions described in the paper it amounts 
to only one-third of a molecular layer of lead peroxide in a 0.001 N solution 
during the course of 1 min. The whole molecular surface layer is replaced only 
after 1 hr has passed. 

In using stable lead peroxide the ideal case of kinetic exchange is much more 
nearly approached — exchange with complete thermodynamic equilibrium 
between the two phases — than when metallic lead is used. 



Originally published in Ber. dtsch. chem. Ges. 53, 410 (1920) 

9. THE INTERMOLECULAR EXCHANGE OF ATOMS 

OF THE SAME KIND 

George Hevesy and Laszlo Zechmeisteb 
From the Chemical Institute of the School of Veterinary Medicine, Budapest 

The present study is intended as a contribution to the answer of tlie 
question as to whether and when interchange of similar atoms takes 
place within a molecule and also between neighbouring molecules of 
like or unlike kinds. In considering a benzene molecule, for example, 
the question arises as to whether a carbon or hydrogen atom can move by 
exchanging places with another similar atom from one position to another 
in the benzene hexagon, or whether a certain hydrogen atom is al- 
ways bound to the same carbon atom. If two neighbouring benzene 
molecules are considered there is the further question as to whether 
carbon or hydrogen atoms which were originally present in the first 
molecule may or may not be found in the second molecule after a 
definite time. 

Such an exchange of positions could be produced either directly by 
the atoms vibrating within a molecule periodically entering into the 
sphere of attraction of another molecule, or indirectly in the following 
way : If there is dissociation such that a hydrogen atom splits off from 
each of two benzene molecules the dynamic nature of the dissociation 
process in which the atom is recaptured yields a 50 per cent chance 
that the hydrogen atom which originally was separated from the first 
molecule will enter the second molecule and thus be subjected to an 
exchange of position. Because dissociation and recombination processes 
take place very rapidly^, even the slightest dissociation in the liquid 
phase, where molecular collisions occur extremely often, will lead to 
such exchange in a short time. 

Although this question cannot be decided by experiment with benzene, 
yet this can be done with lead compounds, for example, by means of 
radioactive methods. It is well known that there are different isotopes 
of lead which can be distinguished easily and with certainty througli 
their radioactive properties, although they exhibit the same chemical 
behaviour. By preparing two different compounds of lead, the one from 

1 M. Le Blanc and K. Schick, Phys. Chem. 46, 213 (1903). 



104 ADVEXTURES IX RADIOISOTOPE EESEAECH 

ordinaiy and the othei' from radioactive lead, it is possible to distinguish 
any atom of lead in the one substance from any atom of lead in the other 
since they have distinct properties. By dissolving the two compounds 
and after a certain time separating them again a simple measurement 
of radioactivity will show whether each atom of lead is still in the same 
kind of molecule as before the experiment or whether an exchange of 
atoms has taken place. 

It has previously been demonstrated^ that Avhen equimolecular 
quantities of inactive lead chloride and active lead nitrate are dissolved 
and the latter subsequently reciystallized half of the active lead atoms 
originally present in the nitrate molecules transfer to the lead chloride. 
The originally inactive lead chloride was proved to be half as radioactive 
after the experiment as the lead nitrate was before. 

The same result was obtained with the following combinations : Lead 
nitrate (active) and lead chloride in pyridine ; lead formate (active) and 
lead acetate in water ; lead acetate (active) and lead tetra-acetate in 
glacial acetic acid ; and lead tetra-acetate (active) and lead acetate in 
glacial acetic acid. 

In contrast it was found that there is no exchange of lead atoms when 
the lead is firmly bound to carbon. The behaviour of organically bound 
lead is illustrated by the following examples : Lead chloride (active) 
and tetraphenyl lead dissolved in pyridine ; lead acetate (active) and 
tetraphenyl lead in amyl alcohol ; and lead nitrate (active) with diphenyl 
lead nitrate in dilute ethyl alcohol. 

The original activity or inactivity of the dissolved substances in these 
instances was not modified by the experiment. The conclusion to be 
drawn from these findings is that exchange of atoms does not take place 
even if the lead is undissociated in only one of the two compounds. 
Exchange is even less likely when this type of binding of the lead exists 
in both components and particularly when these components are che- 
mically identical, in other words, when they are molecules of the same 
substance. There will be no exchange of lead atoms between two mole- 
cules of tetraphenyl lead. The results up-to-date suggest, therefore, that 
an intermolecular exchange of atoms (at least in the time required to 
perforin chemical operations) is connected with the existence of an 
electrolytic dissociation. 

The existing experimental data are inadequate to prove whether two 
similar atoms of the same molecule are able to exchange in a measurable 
time, although it can be assumed probable that the opportunities for 
positional exchange within one molecule are similar to those arising 
between two neighbouring molecules. We intend to attack this problem 
more closely by introducing a radioactive and an inactive lead atom into 

iG. Hevesy and K. Rona, Phys. Cfiem. 89, 303 (1915). 



THE INTERMOLECLLAR EXCHANGE OF ATOMS OF THE SAME KIND 105 

1he same molecule but with different bonding. A further range of appli- 
cation for our method might be opened up by splitting off one of the two 
lead atoms and making (comparative measurements of radioactivity 
on the products. 

EXPERIMENTAL 

The radioactive lead was prepared in the following way : The active 
deposit of a strong radiothorium preparation, which had been recovered 
from a mesothorium, sample whose activity (gamma) corresponded to 
5 mgm of radium, was collected on the surface of a negatively charg(>d 
lead foil. The activated lead was then dissolved in nitric acid and the 
resulting nitrate was converted as required into compounds such as 
chloride, formate, acetate, etc. The salts obtained in this way wer(> 
labelled radioactively with the lead isotope thorium- B. 

The activity was measured in the usual way with the aid of an a- 
electroscope. The substance to be measured was spread on a metallic 
surface and its activity compared with that of a control substance of 
the same weight and identical surface conditions. 

1. Lead Nitrate (Active) and Lead Chloride in Pyridine 

An amount of chloride (0.76 gm) and 0.90 gm of nitrate were dissolved 
completely in 100 gm of boiling pyridine and the solution was kept hot 
for 14 hr. The lead chloride which crystallized on cooling was filtered at 
the pump, washed with a little cold pyridine and with ether to remove 
the solvent, and dried in a vacuum. The sample was quite free from nit- 
rate. Chloride prepared from the original nitrate was used as a stan- 
dard. 

Measurement : 0.268 gm of substance caused the following ionizations : 
Experimental sam})le, 2.76 scale divisions per min (calculated for com- 
plete exchange ^ X 5.64 = 2.82 scale divisions per min). Standaid 
sample, 5.64 scale di\ isions per min. 

2. Lead Formate (Active) and Lead Acetate in Water 

A quantity of lead formate (3.00 gm) and 3.83 gm of su^ar of lead (Kahl- 
baum) were dissolved in 25 ml hot water. After keeping warm for ^/_^ hr 
the majority of the sparingly soluble lead formate was crystallized l)y 
cooling the solution ; the crystals w-ere filtered at the pump, freed 
from traces of adhering acetate by washing with alcohol and dried in 
a vacuum. A sample of the active lead formate was used as a stan- 
dard. Measurement with 0.604 gm of each substance* : Experimental 



1 06 ADVENTURES IN RADIOISOTOPE RESEARCH 

sample, 32.26 scale divisions per min (calculated for complete ex- 
change, 32.43 scale divisions per min) ; standard sample, 64.86 scale 
fli visions per min. 

In a further experiment the standard substance used was the residue 
obtained by evaporating the mother liquor from the precipitated for- 
mate. The accuracy of the experiment was sufficient to establish the 
uniform distribution of the activity between formate and acetate. In this 
experiment 1.00 gm of formate and 1.30 gm of acetate were dissolved in 
20 ml of w^ater. 

Measurement with 0.134 gm of each substance : Experimental sub- 
stance, 22.38 scale divisions per min ; standard 20.61 scale divisions 
per min. 

3. Plumbous Acetate (Active) and Plumbic Acetate in Glacial Acetic Acid 

Plumbous acetate (1.60 gm) was dissolved in a little glacial acetic acid 
and the whole was poured into 50 ml of hot glacial acetic acid containing 
2.20 gm of dissolved crystalline plumbic acetate^. The solution, after 
clarifying by filtration, was kept for 10 min at 80°C, diluted with water 
to four times the volume and boiled. Measurements were made on the 
deposited lead peroxide after washing with dilute acetic acid and al- 
cohol and drying. The residue obtained by evaporating an aliquot 
part of the filtrate was used as standard. 

Measurements were made with 0.136 gm of each substance : Experi- 
mental sample, 20.00 scale divisions per min; standard, 21.95 scale 
divisions per min. 

4. Plumbic Acetate (Active) and Plumbous Acetate in Glacial Acetic Acid 

The active plumbic acetate was prepared by the addition of an active 
sample of red lead, obtained by the oxidation of lead monoxide^, to 
glacial acetic acid. A portion of the beautifully crystalline acetate was 
used as a standard. Plumbic acetate (1.72 gm) and 1.48 gm of plumbous 
acetate (Kahlbaum) were dissolved in 15 gm of hot glacial acetic acid 
to an almost completely clear solution. The plumbic salt which crystallized 
on cooling the solution for some time was washed with cold glacial ace- 
tic acid, and dried by pressing and in a partial vacuum. 

Measurements on 0.400 gm of each substance : Experimental sample, 
1 .29 scale divisions per min (calculated for the case of complete exchange, 
1.43 scale divisions per min) ; standard, 2.86 scale divisions per min. 

1 A. Hutchinson and W. Pollard, Soc 63, 1136 (1S93) : Ibid. 69, 212 (189G). 
See also A. Colson, C. R. Acad. Sci, Paris 136, 670, S91, 1666 (1903). 
-L. Vanino, Praparative Chemie 1, 488 (1913). 



THE IXTEKMOLECULAK KXCKAXGE OF ATOMS OF THE SAME KIM) 1 (jT 

5. Lead Chloride (Active) and Tetraphenyl Lead in Pyridine 

Tetraphenyl lead, Pb(CgH5)4, can be prepared in accordance with the 
<lcscription by P. Pfeiffer and P. Truskier^. The compound can be 
recrystallized from hot pyridine or amyl alcohol. 

Tetraphenyl lead (1.70 gm) and then 0.92 gm of lead chloride were dissol- 
\ (hI in 95 ml of pyridine at the temperature of the boiling water bath 
and, after heating for l^ hr, the solution was cooled to about 35° C. 
A mixture of the two substances crystallized from which the lead chlor- 
ide was extracted by boiling with water. The lead chloride showed the 
same activity as before the experiment. 

Measurement with 0.180 gm of each substance : Lead chloride before 
the experiment, 7.39 scale divisions per min ; lead chloride after the 
experiment, 7.32 scale divisions per min. 

6. Plumbous Acetate (Active) and Tetraphenyl Lead in Amyl Alcohol 

Some acetate (0.70 gm) and 1.00 gm of the tetraphenyl compound were 
dissolved in 70 ml of hot amyl alcohol and the solution w^as kept near 
its boiling point for 15 min. Crystallization of tetraphenyl lead took 
place gradually on cooling and became complete overnight. The filtered 
product was thoroughly washed with amyl alcohol, ethyl alcohol and 
hot water, in succession, and dried in a vacuum desiccator. This sample 
proved to be completely inactive whereas a sample of the lead acetate 
prepared as a standard was very radioactive. 

Measurement with 0.800 gm of each substance: Tetraphenyl lead after 
the experiment, less than 0.02 scale divisions per min ; lead acetate 
(standard substance), 180.0 scale divisions per min. 

7. Lead Nitrate (Active) and Diphenyl Lead Nitrate in Dilute Ethyl Alcohol 

Diphenyl lead nitrate crystallizing with two molecules of water, viz. 
(r6H5)2Pb(N03)2 + 2 HgO, was prepared by the method of A. Polis^ 
by adding tetraphenyl lead to nitric acid. 



1 P. Pfeiffer and P. Truskier, Ber. dtsch. chem. Ges. 37, 1125 (1904) ; cf. 
K. A. Hoffmann and V. Wolfl, Ibid. 40, 2428 (1907). 

'^ A. PoLis, B. 20, 717 (1887). This information was confirmed by P. Pfeiffer 
and P. Truskier, Ber. dtsch. chem. Ges. 37, 1125 (1904). — The observation already 
made by Polis that the formation of diphenyl lead nitrate is disturbed by the 
appearance of dark coloured substances when the hot concentrated nitric acid 
is cooled a Httle below its boiling point is an interesting one. Only the boiling 
acid can therefore effect the smooth course of all intermediate steps since, other- 
wise, the reaction takes a diffcient coiirsc. 



108 ADVENTURES IN RADIOISOTOPE RESEARCH 

A weight of 1 .00 gm of lead nitrate and 1 .59 gni of the diphenyl compound 
wore dissolved in 30 ml of 48 % hot alcohol in the presence of two drops 
of dilute nitric acid. Since there was no deposit within ^ hr, the solution 
was evaporated almost to dryness on a w-ater bath and the crystalline 
mixture taken up in 30 ml of 95 % hot alcohol, lead nitrate then being 
deposited on controlled cooling. This was treated six times with boiling 
absolute alcohol and dried in a vacuum. A sample of the original lead 
nitrate served as the standard sample. 

Measurement with 0.355 gm of each substance : Lead nitrate before 
the experhnent, 5.55 scale divisions per min ; lead nitrate after the 
experiment, 5.80 scale divisions per min. 



S uiuniary 

(1) It has been found that organically bound lead atoms do not undergo 
intemiolecular place exchange in a homogeneous phase. 

(2) Such place exchange occurs to an extent corresponding to that calculated 
from probability when the lead atoms are dissociable. 

(3) Radioacttive indicator methods were employed to obtain these results. 



109 



Comment on papeks 7 — 9 

In paper 7 it was shown that a kinetic interchange takes place between the loud 
atoms of solid lead chloride and the lead ions of a surrounding satnialed lead 
chloride solution. This problem was later studied in detail by Paneth. lie found 
that the uppermost molecular layer of lead sulphate powder participates only 
in an interchange process. When investigating the behaviour of natural crystals 
of lead compounds in several cases he found just a fraction of the lead atoms 
of the uppermost molecular layer participated in a kinetic interchange, piesumably 
positioned at the edges of the crystal. The other extreme case, an interchange 
of almost all atoms of a precipitate with those of the surrounding solution, was 
observed in the case of freshly prepared silver bromide b_\^ Langer and by 
Zimmer. 

In paper 7 the velocity of dissolution and that of intert^iange of massive and 
molecular layers was compared ; among other things, it was demonstrated that 
the velocity of dissolution of ThB is diminished in the presence of lead ions in 
the surrounding solution : however, the velocity of dissolution of the bismuth 
isotope The was not diminished. This investigation, carried out between 1913 and 
1914, aimed at the demonstration of the identity of the behaviour of isotopes. 
\\'hen extending these studies to an interchange between the lead atoms of a 
lead foil and the surrounding lead ions, several hundred atomic layers were found 
1 o be involved in an interchange process presumably due to a dissolution of more 
elec^tropositive parts of the lead foil followed by a precipitation of lead atoms on 
moi"e electronegative parts of the foil. An adsorption of lead ions on the metallic 
sur"face takes place as well, but its role is insignificant compared with the inter- 
change of lead atoms. An investigation of the behaviour of colloidal load particles 
c^arried out by the author and M. Biltz in 1929 brought out a marked adsorption 
of lead ions by the colloidal lead particles and a slow interchange only betweeir 
the lead atoms of the colloidal particles and the lead ions of the liquid phase. 
In a simultaneous investigation of a system composed of colloidal copper and silver 
ions (i.e. two metals showing a marked difference in their electrochemical potential) 
besides some adsorption of silver ions on copper colloids an intense replacement 
of copper atoms by silver atoms was observed. 

In papers 8 and 9 interchange of atoms between heterogenous phast^s \\as 
studied. Paper 9 contains a report on experiment aimed at the elucidation if 
and to what extent interchange of atoms takes place in a homogeneous phase. 
When dissolving in the same solute non-radioactive tetraphen^l lead and labelled 
lead chloride, or vice versa, no interchange of lead atoms was observed; this is 
in contrast to a solution containing 1 mole of non-radioactive lead nitrate and 
1 mole of labelled lead chloride ; after subsequent separation by crystallization 
an equipartition of the radioactive lead atoms was found to lake place between 
tlie chloride and nitrate of lead. The last mentioned result can be consideied 
to be the most direct proof of the theory of electrolytic dissociation. 

Reference 

A. Langer (1943) J Chem. Phy.s. 11. 11 

K. Zimmer (1946) Arhiv f. Kemi, A 21, No 17. 

G. Hev-esy and M. Biltz (1929) Z. Phij-s. Chem. B 3, 271. 



Originally published in Ann. Fhtjs. 65, 216 (1921). 



10. SELF-DIFFUSION IN SOLID LEAD 

J. Groh and G. Hevesy 
From the Chemical Institute of the School of Veterinary Medicine of Budapest 

We have recently shewni that the velocity of self-diffusion, that is, 
the velocity with which the atoms (molecules) of molten lead change 
places, can be ascertained by determining the velocity with which a 
radioactive lead isotope spreads in molten lead. Experiments will now 
be discussed whose purpose is the determination of the self-diffusion 
velocity in solid lead. 

The extraordinarily high resistances which oppose place exchange in 
the solid state led from the outset to the expectation of very slow self- 
diffusion in soUd lead ; we have, therefore, avoided setting up experiments 
at room temperature and have sought rather to determine the self- 
diffusion in lead heated and maintained about 40 °C below its melting 

point. 

Several series of experiments lasting from 1 to 3 months showed that 
the self-diffusion velocity of lead at 280°C, that is, 46° below its melting 
point, is less than 0.001 cm^/day. A series of experiments was then 
performed in which lead filaments, about 2 cm long, were heated for 
more than 400 days ; these filaments consisted, as will be described 
in detail below, of a 1 .5 cm long inactive and a 0.5 cm long active portion 
of lead. No diffusion of the active lead isotope into the inactive lead 
could be detected even after this long period of experiment. The self- 
diffusion constant of the solid lead is accordingly still smaller than 
0.0001 cm2/day, even at a temperature of 280°, since values of this order 
could still have been easily determined in the stated conditions. 

This result is not without interest, especially when it is compared 
with the well-known Roberts-Austen experiments^. Roberts-Austen 
allowed gold to diffuse into solid lead and found the diffusion constants 
recorded in the table below, which also includes our experimental result. 

Even at 251°C, therefore, the diffusion of gold into lead is at least 
three thousand times as fast as that of lead into lead at 280°C, the 

ij. Groh and G. Hevesy, Ann. Phys. 63, 85 (1920). 
2 W. C. Roberts-Austen, Phil. Trans. 187, 404 (1896). 



SELK-niKKlSIOX IX SOLID LEAD 



111 



T 


Gold in lead D 


Lead in lead D 


CO) 


(cm«/day) 


(cm/*day) 


100 


U.0U002 





165 


0.0045 


— 


200 


0.0075 


— 


251 


0.026 


— 


280 


— 


< 0.0001 



latter temperature being somewhat higher and thus more favourable 
to diffusion. At this latter temperature, which is not very far removed 
from the melting point, the self-diffusion is still extraordinarily slow 
and should be incomparably slower, for example, at room temperature. 
When relating this result to the velocity of self-diffusion in other metals 
it must be borne in mind that lead is one of the softest metals and that 
self-diffusion should presumably prove to be much slower in the harder 
metals . 

Quantitative data on the diffusion in solid metals have been provided 
only by Roberts-Austen, but metallurgy is plentifully supplied with 
qualitative experiences which point out the relatively rapid diffusion 
of alloying solid materials, of which the rapid interpenetration of iron 
and carbon! supplies at 250 °C the best-known example. Thus there 
exists a very considerable difference between the diffusion of two solid 
metals into each other and the self- diffusion in a solid metal, in complete 
contrast to the diffusion in the liquid media. Thus, we have obtained- 
a value for the self-diffusion constant of molten lead which is only 
slightly different from the constant for gold in lead. 

The main reason why self-diffusion in lead is so much slower than 
the diffusion of gold into lead appears to be that the gold diffusing into 
the lead loosens up the crystal structure and in this way facilitates its 
own transmission. It is found that the introduction of an impurity into 
the crystalline structure can have exactly the same effect as a rise of 
temperature in facilitating the place exchange of the ions (atoms, 
molecules). 

However, it is not stated absolutely that all other metals diffuse more 
easily into lead than do its own atoms ; w^e attempted to allow simultane- 
ous diffusion of the lead isotope radium-D and polonium (Avhich is 
a homologue of tellurium) into lead, but no positive result was obtained. 

Diffusion experiments in solid bodies claim so much interest, for this 
and other reasons, because information can be obtained from the results 
concerning the magnitude of the resistance opposing the displacement 
of individual atoms in the crystal structure. Diffusion experiments of 



iM. A. CoLSON, Ann. Chim. et Phys. 17, 221 (1846). 
2 J. Groh and G. Hevesy, Ann. Fhys. 63, 85 (1920). 



^12 ADVENTURES I\ RADIOISOTOPE RESEARCH 

the Roberts- Austen type, however, are not suitable for obtaining 
the desired information on this point. If it were required to decide upon 
the slowness of place exchange in solid lead from the Roberts-Austen 
data a completely erroneous result would be given, whereas the appli- 
cation of radioactive indicators, i.e. the measurement of the velocity of 
diffusion of a lead isotope in lead yields the required information. 



EXPERIMENTAL METHOD 

The layers of active and inactive lead were joined together by the 
method already describedi. The inactive lead was melted in a vacuum 
in one limb of the Y-shaped hard glass tube and, after it had solidified, the 
fused active lead contained in the other limb was poured on, thereby pro- 
ducing a cohesive metallic cylinder. While the active material used in the 
determination of the velocity of self- diffusion in molten lead was ordinary 
lead labelled with ThB, this procedure was no longer admissible in the 
present instance because the ThB decays with a half-life of 10.6 hr and 
the time of experiment amounted to more than 1 year. Joachimsthal 
lead, a mixture of ordinary lead, uranium-lead and RaD, has therefore 
been chosen as the active material. Of these three lead isotopes only 
the RaD is active and this only to such a small extent that its radiation 
is not suitable for determining the amount of RaD present; the a- 
rays of its daughter product, polonium, however, serve as a convenient 
means for determining the RaD. 

Another point in which the experimental method followed here 
differed from that used for diffusion in the liquid was that, after joining 
together the layers of active and inactive lead, the boundary surfaces 
were fused together by means of a finely pointed flame in order to obtain 
complete contact between the two kinds of lead, this being clearly of 
great importance for uninterrupted diffusion. A completely cohesive 
column was thus obtained but of course mixing of the sharp boundaries 
of the active and inactive lead was unavoidable. In order to take account 
of this fact, we proceeded as follows : 

The column of lead, moulded in the manner described, was cut into 
two vertical sections with a toothed saw and one of these strips was 
sealed in an evacuated glass tube which was then placed into an electrical 
resistance furnace. After the experiment the strip was sectioned at three 
places marked with India ink and was thus separated into four equal 
parts. The second vertical strip had already been cut at the correspond- 
ing places before the experiment and was used as a control. If the active 
layer of lead is denoted by I, then layer II likewise showed some activity 

1 J. Groh and G. Hevesy, Ann. Pliys. 63, 85 (1920). 



SKJ,1'-I)IK1 rsiON IN SOLID LKAl) 1 [;} 

on account of llu> mixing at the boundary; this activity, found even 
l)efore the dil'l'usit)n, could, however, be subtracted from that found after 
diffusion and the mixing at Ihe boundary could be taken into account 
in this way. Yet the correction described would onlv acquire importance 
in the case of an experiment giving a positive result ; since no activity 
noticeably in excess of the natural decay was found in layers III and IV, 
it was unimportant. 

In order to be abl(> to measure accurately the a-activit}- of tli(> in- 
dividual sections we have also used here, as in the experiments which 
served for determining the self-diffusion in molten lead, rolled sheets 
of lead and measured the activity of the disk thus obtained in the a- 
electroscope. The total length of filament amounted to 16 --20 mm. 

The a-activity of polonium indicated by the electroscope is a measure 
of the amount of the lead isotope (RaD) present only if the RaD and 
Po exist in radioactive equilibrium. The amount of RaD which diffused 
in the first four months harl come to more than 80 per cent radioactive 
equilibrium when 14 months had elapsed; that which diffused in the 
second 4 months had reached over 50 per cent after the same time, when 
the measurements were made. The absence of any appreciable activity 
in the layers III and IV made it possible to determine the diffusion 
constants of both lead in lead and polonium in lead at 280 °C as being 
less than 0.0001 cm^/day, without awaiting exactly the establishment o\' 
the radioactive equilibrium between RaD and Po. It is intended, how- 
ever, to follow this process further during the next year and thus to be 
able to extend the observations beyond the determined limits of the 
diffusion constants mentioned above. We are also concerned in work- 
ing out other types of method which permit the determination of very 
much smaller diffusion constants than those mentioned. 

We may mention here that tw^elve lead filaments have been prepared 
as described above, and have been introduced separately into evacuated 
glass tubes and heated in an electric resistance furnace for 400 days. 
The furnace temperature, which varied between 270 and 290°C', was 
followed constantly with a quartz thermometer, since our experience 
with continuously heated glass thermometers in similar experiments 
has been unsatisfactory. 

Summary 

The self- diffusion velocity of solid lead has been d(>t<Mniined by following the 
diffusion of the lead isotope radium- D in solid lead at 280° (' for more than 1 year. 
The diffusion constant, even at this temperature which is only 4()'' helow the 
melting point, is shown to be still less than 0.0001 cm^/day. Self -diffusion in l(^ad 
thus takes place at least two-hundred times more slowly than the diffusion of 
gold in solid lead at the same tempeiatuie. 



8 He 



vesy 



Originally published in Xattirc, 115, <)74 (1925) 



11. SELF-DIFFUSION IN SOLID METALS 

G. Hevesy and A. Obrutsheva 
From tho Institute of Theoretical Physics, University of Copenhagen 

The "sagacity" with which atoms, or groups of atoms, oscillating about 
fixed points in the crystal lattice, refuse to exchange position with 
neighbouring atoms, is often regarded as one of the chief characteristics 
of the crystalline state. On the other hand, numerous cases are recorded 
in which crystalline bodies, for example, solid metals, penetrate into 
each other, in which, therefore, a replacement of the atoms of one metal 
by those of the other takes place. The classical experiments of Roberts- 
Austen on the diffusion of gold in lead bars are widely known. At a 
temperature as low as 100° he found the diffusion coefficient of gold 
in lead to be 2 x 10"* cm^ day-i, being thus only about 100,000 times 
smaller than that of sodium chloride in water. Several cases of inter- 
penetration of solid metals have been recorded since, including the 
interesting case of the diffusion of thorium in heated tungsten wires, 
reported recently by Langmuir. But it must be noticed that from the 
rate at which one metal like gold diffuses in another like lead, no con- 
clusion can be drawn about the velocity with Avhich the atoms change 
their position either in a bar of pure lead or of pure gold ; no conclusion 
can be drawn on the rate of self-diffusion in these elements. 

The idea of self- diffusion was introduced by Maxwell, when calculat- 
ing the rate of diffusion of gases. The calculation was very much simplified 
by considering the case in which the molecules of the two diffusing 
gases had the same properties, for example, the exchange of place of 
molecules in a column of nitrogen. The use of the radioactive isotopes 
of lead enabled one of the writers, in collaboration with J. Groh (Ann. 
(I. Phys. 65, 216 [1921]) to realise a measurement of self-diffusion in 
the case of liquid and solid lead, the diffusion in liquids and solids being 
practically independent of the difference in the masses of the isotopes. 
For the rate of the self-diffusion in molten lead, namely, of thorium B 
in molten lead, close to the melting point, the value found was 2 cm- 
day-^. In the solid metal, however, after heating a bar, the upper pari 
of which was composed of radio-lead, for about a year at 280°, and 
llien analysing the lower part with the electroscope, no diffusion could 



SKJ.F-BIFFUSION IN SOLID METALS 115 

1)0 Ibund. It was, therefore, concluded that Ihe sell'-dif'fLusion in solid 
lead is, even at this high temperature, less Ihan lO""* cm^ day~i. 

To increase the sensitiveness of the method, we prepared in the present 
work two thin foils, one of ordinary lead. Hie oilier wilh lead containing 
thorium B in homogeneous mixture, and pressed these together in 
vacuo. The thickness of the inactive foil was chosen slightly greater than 
the range of the a-particles to be measured ; therefore no scintillations 
originating from the radioactive lead could be observed wdien investigat- 
ing the inactive foil. But, on heating the aggregate of the foils, a diffusion 
of the active lead into the inactive one took place and the a-particles 
due to the diffused atoms or their successive products of disintegration 
produced scintillations on the observing screen. By comparing the number 
of these scintillations with the number of scintillations produced by 
the active foil at the beginning of the experiment, the rate of self- 
diffusion in lead was determined. The following values were found : 



t" 


D 


ill em- Jay 


l' It in cm^ day~ 


260° 




6 X 10-7 


310° 5.7 X 10-6 


280° 




1.5 X 10- « 


320° 4.7 X 10-5 


300° 




2.5 X 10-6 


324° 1.4 X 10-* 



The diffusion rate 2° below the melting point is thus 10,000 times smal- 
ler than in molten lead. 

When investigating the diffusion of two very similar metals like silver 
and gold, or thallium and lead, into each other, we can expect to find 
conditions not very far removed from those encountered in the case of 
self-diffusion. By using a foil of thallium and one of active lead it was 
found that the coefficient of diffusion of lead in thallium amounts at 285°, 
i.e. 15° under the melting point of the latter, to 2 x 10-^ cm^ day-^. 

On the other hand, when investigating the diffusion of two not simi- 
lar metals into each other, much more intricate conditions were to be 
expected. We determined the rate of diffusion of polonium, which is 
the highest homologue of sulphur, into both lead foils and single crystals. 
The coefficient was found about the same both in the foil and crystal 
(at 310° Z) = 1.3 X 10-5 eni2 clay-i). In this connexion it may be men- 
tioned that, in discussing the discrepancy between the values of the 
period of decay of polonium found by different investigators, Mme. Cu- 
rie has put forward the explanation, that during the long time of 
observation, the polonium in some cases diffused into the metal from 
the surface of which it was collected. Recently, Maracineanu (C i?. 
176. 1879. (1923)), working in Mme. Curie's laboratory, has obtained 
evidence that the apparent period of polonium is appreciably shorter 
if the lead on which it is collected is heated for a while. 

8* 



Originally published in Z. Phijs. 79. 197 (1932) 

12. THE HEAT OF RELAXATION OF THE LEAD 

LATTICE 

G. Hevesy, W. Seith and A. Keil 
From the Institute of Physical Chemistry, University- of Freiburg 

The heat of relaxation of the lead lattice (the work of release of the lead 
atoms) is determined from the temperature coefficient of the velocity of 
diffusion of a radioactive lead isotope in lead ; a study is made of the 
sensitivity to structure of this quantity and the velocity of diffusion. 
Besides the heat of vaporization, heat of fusion and lattice energy 
there is another quantity of energy which is important to the crystalline 
state of aggregation. This is the heat of relaxation of the crystal lattice, 
or the energy of release of the lattice components. The latter is the energy 
of activation of self- reaction, which takes place between the atoms of 
a metal and results in place exchange of the lattice units. ^Vhen the 
transport number is known the heat of relaxation of ions of an electro- 
lytic conductor can be calculated from the temperature coefficient of 
the conductivity. Direct measurement of the velocity of self-diffusion 
is the only course open in dealing with metals. The heat of relaxation, 
Q, is calculated from the diffusion constant, Z), measured at various 
temperatures, by using the well-known formula 

D = .4e-<? '^^ 

where J. is a constant which is practically independent of the tempera- 
ture. In the measurement of self-diffusion it is usually necessary to follow 
the speed of mixing of two closely related metals, such as gold and 
silver or tungsten and molybdenum, which is then equated as a first 
rough approximation to the speed of self-mixing of one component. 
The velocity of self-diffusion in lead can be determined accurately, 
without such an uncertainty, by measuring the velocity of diffusion of 
a radioactive lead isotope in ordinary lead. Pievious experiments of 
this kind have already been described^. 

This paper will deal with the result of an investigation carried out 
recently with the object of determining the heat of relaxation of the 
lead lattice and of ascertaining how far this quantity and the velocity 

^ G. Hevesy and A. Obrutschew.^, Nature 115, (174 (1925). 



THE HEAT Ol' KELAXATION OK ITIK LEAD LATTICE 117 

ol self'-dilTusion in lead are structure sensitive. The method used lias 
been described in detail on a previous occasion^ TIk^ lead isotope ThH 
is condensed on a lead surface and 1h(> ionization caused by the a-radia- 
tion of the radioisotope (or its decay products) is measured before and 
after the course of dilfusion. The deeper the thorium-B penetrates by 
diffusion into the lead, the greater is the absorption of the a-radiation 
and the smaller will be the ionization arising from it. The calculation 
of the velocity of diffusion of thorium-B in lead, which is the same as 
the velocity of self-diffusion of lead, is executed by means ol' a formula 
developed by R. Furth'^, which correlates the ionization values before 
and after dilfusion. the range of the u- radiation in lead and the lime. 
In a more sensitive modification of the method which has also been 
described the recoil yield, instead of the ionization due to the a-radia- 
tion, is measured before and after diffusion. 

Both "Kahlbaum" lead and lead of very high purity, which was kindly 
made available by the Akkumulatoren-Fabrik A. G., Jiagen, West- 
phalia, were used in the experiments. The lead was freed from its content 
of gases by prolonged fusion in a vacuum and was purified from oxide 
content by passing it through a capillary system. The lead single crystals 
were prepared by the method of Ivyropoulos and were characterized, 
as compared with crystalline lead, by their remarkable resistance to 
oxidation by the air. All the experiments described below were carried 
out with complete exclusion of air either in an atmosphere of nitrogen 
or in a vacuum, and the small tubes containing the single crystals were 
broken in the evacuated apparatus. All the results recorded in Table 1 
can be represented by the equation : 

or Z) = 5.76 X 1()5q-uo25'T 

log Z) = 5.76 — 04343 (14025/T) 

or by a straight line (Fig. 1). Thus Q amounts to 14025 E = 27870 
cal/mole and A = 5.76 x 10^ and, within the limits of experimental 
error, there is no difference lietween the behaviour of the single crystals 
and the crystalline material. 

An investigation was then made as to whether destruction of the 
texture at the surface of the single crystal by a milling machine, with 
the specimen necessarily being exposed to the air for a short time, has 
a measurable effect on the self-diffusion constant. No marked effect 
on the velocity of diffusion due to this manipulation could be found. 
Table 2 shows the results of these measurements. The values thus 
obtained can also be represented by the above equation and by the 
straight line applying to the imworked material. 

1 G. HKVE.SY and W. Seith, Z. Phi/s. 56, 790 (1929): Jhid. 57, 809 (1929). 

2 R. FtJRTH, Handb. d. pht/.s: u. techn. Mechanik 7, 687 (1930). 



118 



ADVEXTUKES IX RADIOISOTOPE KESEAKC'H 



Table 1. — SELF-DirrrsioN in Lead Single Crystals Diffusion 

CONSTANT (D) OF TllB IN LeAD (SELF-DirFL'SION CONSTANT OF LeAD) 



No. 


t 
(°C) 


(cm*/day) 


(i/r)io« 


log7> 


Remarksi 


1 


182 


4.12 • 10-8 


2197 


—7.39 




2 


196 


5.7 • 10-8 


2131 


—7.24 




3 


207 


8.2 • 10-8 


2083 


—7.09 


Crystalline lead 


4 


222 


2.45 • 10-7 


2020 


—6.6 


Single crystal from 


5 


238 


7.3 • 10-' 


1956 


—6.1 


the melt 


6 


245 


9.6 • 10-7 


1930 


—6.0 




7 


245 


6.57 • 10-7 


1930 


—6.18 


Single crystal 
from the melt 


8 


258 


1.1 • 10- « 


1883 


—5.96 




9 


259 


3.4 • 10- « 


1879 


-5.47 




10 


263 


2.3 • 10-6 


1865 


—5.64 




11 


275 


6.0 • 10-6 


1824 


—5.22 




12 


290 


7.2 • 10-6 


1776 


—5.15 




13 


301 


1.6 • 10-5 


1742 


—4.79 




14 


312 


1.62 • 10-5 


1709 


—4.79 




15 


317 


2.82 • 10-5 


1695 


—4.55 


Single crystal 
from the melt 


16 


322 


2.36 • 10-5 


1680 


—4.63 


Crystalline lead 


17 


324 


4.78 • 10-5 


1674 


—4.32 





1 In all cases where there is no remark single crystals grown by the K^-l■opoulos 
method were used ; in items 6, 8 and 13 these consisted of Kahlbaum lead and 
in all other items of lead from the Akkumulatorfabrik, Hagen, ^^'estphalia. 

Table 2. — Diffl'sion Constant of ThB in Cold-Worked Lead 



Xo. 


(h;) 


(cm^/dajO 


(1/2')1U« 


log/* 


liemarks 


18 


196 


4.13 • 10-8 


2130 


—7.38 


Milled single crysf al 


19 


217 


2.87 • 10-7 


2040 


—6.54 


Milled single 


20 


233 


4.57 • 10-7 


1976 


—6.34 


Milled crystallite 


21 


237 


6.75 • 10-7 


1960 


— 6.17 


Milled single crystal 


22 


254 


2.99 • 10-6 


1897 


—5.64 


Milled crystallite 


23 


270 


4.57 • 10-6 


1841 


—5.34 


Tempered crystallite 



The results discussed above relate to a temperature range which 
extends from the melting point of lead (327°) to 182°. Below the latter 
temperature the self-diffusion can be followed by making use of the 
recoil effect. As has already been mentioned, this method does not 
make use of the decrease in ionization after diffusion but the recoil 
yield is determined. While the range of this a-radiation in lead is 3 X 10-^ 
cm the range of the recoil particles extends only to a thickness of about 
one hundred atom layers (4.7 x 10-® cm). In corresponding degree to 



THE HEAT OF JJELAXATION Ol' THE EKAD LATI rf'K 



119 



the shorter range of the recoil particles the latter method is indeed 
considerably more sensitive than the method first described. In the 
study of the self-diffusion of lead ions in lead iodide it has been found 
possible to determine that the measurement of a-radiation and recoil 
yield the same result ; in spite of the fact that the recoil measurements 
register processes in th(> top few hundred molecular layers they were 




28 27 26 2S 2^ 23 22 21 20 19 IS 17 

^T'lO^ 

Fig. 1. Rate of Diffusion of labeUed solid Lead. Schmelztemperatui — 

Melting point. 



Iff 



as highly reproducible as the a -measurements. Recoil measurements at 
lead surfaces, on the contrary, indicated a high sensitivity of the metallic 
surface to external effects. For example, contact of the lead sampk 
with air for a short time was sufficient to lower the diffusion values 
and even the values obtained in a carefully purified nitrogen atmos- 
phere were rather lower than those determined when working in a 
vacuum. In spite of the uncertainty arising for these reasons in the 
recoil values, the experimental points obtained by this method also 
lie approximately on the straight lines obtained with thea-measurements 
as the basis (cf. Fig. 1) ; it must also be borne in mind that the recoil 
range in lead is not accurately known and that it must be calculated 
by extrapolation from the values measured in air. 

The temperature coefficient of the velocity of diffusion is in quite 
good agreement with the results of the already mentioned preliminary 
experiments, but the difference in behaviour of single crystals and crys- 



12(1 



ADVENTUJRES IN BADIOISOTOPE KESEARCH 



Table 3. — Diffusion Constant (D) of Thoriitm B in 
Lkad Determined by the Reooil Method 



>:o. 


t 
(°C) 


D 
(cmVday) 


(i/r)io« 


logK 


Kemarks on heating 
of the lead 


1 


lOfi 


1.4.-, • 10-11 


2640 


10.84 


Nitrogen 


2 


113 


1.42 • 10-- 11 


2.590 


9.85 


Vacuum 


3 


114 


2.60 • 10-11 


2.584 


— 10..59 


Nitrogen 


4 


120 


1.93 • 10-10 


2.544 


— 9.72 


Vacuum 


ij 


128 


2.3.5 • 10-10 


2494 


— 9.63 


Vacuum 


6 


129 


1.97 • 10-10 


2487 


— 9.71 


Vacuum 


7 


136 


3.41 • 10-10 


2444 


— 9.47 


Nitrogen 


8 


137 


4.59 • 10-10 


2438 


— 9.34 


Vacuum 


9 


137 


3.41 • 10-10 


2438 


— 9.47 


Nitrogen 


10 


141 


l.OS • 10-10 


2415 


— 9.97 


Vacuum 


11 


144 


3.21 • 10-10 


2397 


— 9.49 


Vacuum 


12 


153 


3.29 • 10-10 


2346 


— 9.48 


Nitrogen 



lallites demonstrated in those experiments could not be reproduced, 
possibly because the single crystals in the preliminary experiments 
were unavoidably exposed for a long time to contact with the air (during 
counting of the scintillations). The present study shows much more 
pointedly that neither the heat of relaxation of the lead lattice nor the 
velocity of self-diffusion of lead atoms is structure-sensitive. This result 
is possibly connected with the ready recrystallizability of lead since in 
the molybdenum — tungsten system van LiemptI was unable to find a 
structure dependence of Q, yet A and therefore the diffusion constant 
showed such dependence. He found A to be about eight times as large 
in polycrystalline material as in a single crystal, by measuring the veloc- 
ity of diffusion of molybdenum in tungsten, and even earlier a structure 
sensitivity of the electrolytic conductivity, which is closely related to 
the self-diffusion, had been demonstrated in salts^. 

VAN LiEMPT calculates the constant A from the equation 

A = n .r2 r/6 

^\here x is the distance l)etween lattice planes and v the vibrational 
frequency of the atom. In the case of molybdenum diffusing in tungsten 
single crystals he finds remarkably good agreement between the observed 
and calculated values of A. The value of A which we have measurerl 
in lead is, on the contrary, about one thousand times the value calculated 



IS. A. M. VAN LiEMPT, Z. (tnorg. diem. 195, 3GG (1931): Rec. Trav. Chim. 
51, 114 (1932). 

2G. Hevesy, Z. Phys. 10, 80 (1922) : G. Tammann and G. Veszi, Z. anorg. 
Chcm. 150, 355 (1926) ; T. E. Phipps, W. D. Lansing and T. G. Cooke, J. Amcr. 
(hem. Soc. 48, 112 (1926), eiv. 



THE HEAT OF RKL.VXATIO.V OF THK LEAD LATTICE 



121 



by VAN Liempt's method. Tlio s 'irdilfiision (s:-lf-ivaction) in lead accord- 
ingly represents, at least formally, an example of a chain reaction in 
which the chain length is independent of whether single crystals oi' 
polyerystalline materials ar(> involved. 

The magnitude of the heat of relaxation is compared, in Tal)le 4. with 
the energ}^ content and the heats of fusion and vaporization. 

T.\i!LK 4. — TTk.\t Pk()Pp:kties of Le.\d 



kc:il/K iitoni 



Heat of fusion 

Energy content at the melting point 

Heat of relaxation 

Heat of vaporization 



1.1 
3.5 

27.9 
36.2 



Table 4 shows clearly that the heat of relaxation is not very mudt 
less than the heat of vaporization but that it is very much greater than 
the energy content at the melting point and the heat of fusion. 



Suinniary 

The heat of relaxation of the lead lattice (heat of activation for the self-reaction 
of the lead atoms) amounts to 27,830 cal/mole. This quantity, like the constant 
A of the diffusion equation, is only slightly stiuctuie sensitive. 



Originally published in Z. f. Elektrochem. 37, 52S (1931 



13. DIFFUSION IN METALS 

G. Hevesy and W. Seith 
From the Institute of Physical Chemistry, Uni\ersity of Freiburg 

Diffusion in salt-like compounds is facilitated by means of a relaxation 
process which occurs when the crystal is heated. This relaxation which 
exhibits some similarity to activation in the theory of reaction velocity 
depends chiefly on the size, valency, electron affinity and polarization 
properties of the lattice components. In silver iodide, for example, 
where the small univalent strongly-polarizing silver ion contrasts with the 
large iodide ion which has slight attraction for electrons and is easily 
polarizable, there is easy detachment of the silver ion and it is well- 
known that mobilities indeed exceed those occurring in aqueous solutions. 
In the pure metal the behaviour is altogether different. In such case 
there is only one kind of lattice component, a high co-ordination number 
and a high symmetry of charge distribution. Large diffusion velocities 
cannot therefore be expected in pure metals. Metal alloys are different. 
In the lead-gold system, for example, the small gold atom which has a 
high affinity for the valence electron contrasts with the larger lead atom 
which has a lower electron affinity, and hence there occurs a system which 
is readily subject to relaxation in which the gold atom easily vacates 
its position. Roberts- Austen in his classical investigations has 
already been able to demonstrate that gold diffuses into lead even at 
moderate temperatures with a considerable velocity. The velocity of 
diffusion of gold in lead is attained through the speed of dissolution of 
gold atoms in the gold-lead phase and in its taking up a new position. 
The gold penetrates into lead but, on the contrary, lead is practically 
immobile in gold. The diffusion constant for gold in lead is 4x10^3 
cm^ day~i at 150° whereas the diffusion of lead in gold-lead at a temper- 
ature of 141° amounts only to SxlO^^^ cm^ day^^, i.e. it is smaller by 
seven orders of magnitude. Lead atoms accommodated near silver atoms 
are more easily dissolved than those considered above. It has been 
found that lead diffuses about twice as quickly in silver saturated with 
lead as in pure lead. 

If the gold in lead alloys is replaced by other elements whose properties 
become more and more similar to those of lead, then these elements show a 



DIFFUSION IX METALS 



123 



stoadily decreasing diffusion velocity. The margin l)etween the diffusion 
velocity of the added element and that of lead thus becomes steadily 
smaller and the unilateral fliffusion becomes gradually less apparent. 
Considering now an alloy of ordinary and radioactive lead, both con- 
stituents will exhibit the same diffusion velocity. This is an example of 
self-diffusion and therefore a complete mutual replaceability in the 



220 2^0 260 2S0 300 320 




Fig. 1 . Diffusion in Blcilogieiungen — Diffusion in lead allo,>s 
Schmclzpunkt des Blcis — Melting point of lead 



diffusion process. The gradual decrease of diffusion velocities of metals 
in lead in the sequence, Au — Pb, Pb — Pb, and also the step- wise increase 
of the heat of relaxation (heat of activation), can be seen in Fig. 1 . The 
latter quantity is also recorded in Table 1. 

The marked preferential diffusion of one component is also encountered 
in salt-like compounds but there is no mutual replaceability of the 
components which, indeed, is a characteristic of metallic systems. The 
silver in silver chloride can only change places with silver; it is other- 
wise in metallic alloys. Considering the place-exchange processes in a 
saturated silver-lead phase, the readily detachable silver atom will leave 
its place with great frequency in unit time. The silver atoms intermit- 
tently seek out the lead atoms and occasionally also replace other silver 



124 



ADVENTURES IN EADIOISOTOPE EESEARCH 



Table 1 



Metal 


ij •-'.jd 

(cm* day—') 


'J 
fcal) 


Au 


3 • 10-2 


--14,000 


Ag 


2.6 • 10-3 


15,000 


Bi 


3.2 • 10-5 


18,500 


Tl 


1.9 • 10-5 


18,500 


Sn 


4.4 • 10-6 


c. 28,000 


Pb 


1.3 • 10-« 


c. 30,000 



atoms. The lead atoms on the contrary scarcely ever leave their places. 
At room temperature, a lead atom in pure lead changes its place once 
in 10 days while the silver atom in lead-silver alloy vacates its position 
100 times per sec.^ As the temperature is raised the difference diminishes. 
\Ve have already been able to demonstrate, in an earlier paper^, in the 
case of silver ahoys that the speed of place exchange of the atomsde- 
creases as the ideal metallic state is approached. The last step, however, 
i.e. the measurement of self-diffusion in silver, was not practicable in 
the systems mentioned. We have therefore applied our attention chiefly 
to lead alloys because they presented an opportunity for determining 
also the self-diffusion in lead. 

In determining the frequently very low velocity of diffusion, use was 
made of quantitative optical-spectroscopic analysis which has been 
found especially suitable for this purpose. Indeed this methods permits 
both the determination of very small amounts of metal and the use of 
very thin and strictly localized layers in the analysis. Samples of known 
composition are first prepared, e.g., lead-thallium alloys with the 
concentration varying between 3 and 0.01 per cent and the ratio of the 
intensities of suitable lines of the two elements is determined. Layers, 
each 0.05 mm thick, are cut after diffusion and these are analysed 
spectroscopically by comparing with the test samples in accordance 
with the method described by Gerlach^. The diffusion constant can be 
calculated since the concentration, as a function of the distance from the 
original boundary, is known. In the method described it is above all 
necessary to ensure that the metal whose diffusion constant is to be 
determined is distributed as atoms in the lead. If this does not appl} 
1hen the analytically determined concentration is not identical Avith 



1 Concerning the calculation of the frequency of place exchange of an indi\i(lual 
atom, refer to H. Braune, Z. phys. Chem. 110, 147 (1924) ; J. Frenkel, Z. Fhys. 
35, 652 (1926) : J. A. M. van Liempt, Z. anorg. Chem. 195, 366 (1931). 

2G. Hevesv, Z. Elektrochem. 34. 463 (1928). 

3 W. Gerlach and E. Schweitzer, Chemical Emission-Spectrum Analysis, 
Leipzig (1930). More details of the application of this method to measurements 
of diffusion will shortly he described bv Guenther and Laird. 



UIFFLSIU-N l-\ .METALS ] 2"^ 

Ihc eonceniration considorcd in the dilfusiou process^. 'Jlic silver-lead 
and gold-lead systems provide examples of the behaviour just referred to. 
►Starting with a coneentrated silver-lead alloy, in which the silver is as 
finely divided as possible, then the silver which has migrated by diffusion 
will constantly be replaced by dissolution from the grains of silver. The 
system therefore consists of a constantly saturated solution of silver in 
lead from which silver diffuses into pure lead. As will be seen later, ihe 
solubility of silver in lead can l)e determined 1)y means of this behaviour. 

In only a few cases have wv us(hI an alternative to the spectroscopic; 
method. The silver content of lead alloys has been determined, for 
example, by Ihe usual method of volatilizing the l(>ud and assaying Ihe 
residual lead of silver. It was also necessary to employ radioactive 
methods in order to determine the self-diffusion rate. At higher temper- 
atures, where the selfdiffusion is already somewhat larger, the diffusion 
constant could be determined as follows : A radioactive lead isotope is 
condensed on the surface of inactive lead and the ionization due to 1he 
a-])articles emitted by the radioactive lead is measured. The system is 
then heated to the experimental temperature and the ionization, which 
now has a lower value owing to the diffusion which has taken place, is 
measured again. The self-diffusion constant can be determined from this 
decrease due to diffusion. At a lower temperature this method, which 
indeed is very sensitive, proved not sensitive enough and had to be 
replaced by another. The recoil yield before and after diffusion was 
measured and the diffusion constant was calculated from the decrease 
by means of a formula developed by R. Furth-. While the first method 
enables the diffusion constant to be determined down to 10~'^ cm^/day. 
the use of the second method permits diffusion constants of 10-^^ cm^ day 
or less to be obtained. 

The solubility of a metal in a solid phase can be determined from th(> 
analysis of diffusion since only those particles of the metal which are 
distributed as atoms are involved in the diffusion process. The dis- 
fussion is best illustrated by an example. 

(a) Starting with a concentrated alloy the diffusion is allowed to 
proceed until all parts of the originally pure layer of lead are saturated 
with the diffusing metal. Since an increase in the diffusion time is no 
longer accompanied by an increase in concentration there now exists a 
saturated solution the analysis of which yields the solubility directly. 
At 288 °C', for example, it is impossible to produce by diffusion a silver- 
lead alloy containing more than 0.13 atomic per cent of silver. 

1 ff. G. Grube (Z. Metallk. 19, 438 [1927]), who has (Iclcrminod a scmmp.s of 
diffusion velocities in high-melting metals. With regard to the problem of diffusion 
in alloys refer also to the many puV)lieations of G. Tammann and his scliool. 

2 R. FtJRTH, Hamlbuch dcr pliy.sikal. and techn. Mechanik 7, <)S7 (1930). 



1 26 ADVENTURES IX RADIOISOTOPE RESEARCH 

This method, of course, requires long experimental times and a time of 
3 months is needed for homogenizing a 5 mm layer even with silver 
Avhich diffuses comparatively rapidly in lead. We have therefore often 
used the following consideration for determining the solubility : 

(b) Disregarding the initial layer (lead-silver alloy) at first, the silver 
concentration is determined at various positions after diffusion in the 
originally pure lead and the diffusion constant is calculated from these 
results. Now if the diffusion constant is known the corresponding con- 
centration of silver can be calculated and this in turn yields the solubility 
of silver in lead. 

Starting with a silver-lead alloy of 

10 atomic per cent 

1 atomic per cent 

0.5 atomic per cent 

we obtained the following values for the solubility of silver in lead 

0.15 atomic per cent 
0.12 atomic per cent 
0.13 atomic per cent 

Hence, starting with silver-lead alloys of various concentrations, from 
which the same solubility values have been obtained, it may also be 
concluded that the silver from the macroscopic grains of silver was supp- 
lemented so rapidly that the original lead-silver alloy always remained 
saturated. If a diffusion experiment is performed, commencing with a 
0.1 atomic per cent silver-lead alloy, the same diffusion coefficient found 
by the method described above is obtained both from the concentration 
of the silver in the initially pure lead layers and from the concentration 
decrease of the silver in the original silver-lead alloy. The method descri- 
bed for determining the solubility is important in so far as there are at 
present no other methods available for determining very low solubilities 
of one metal in another^. 

Summary 

The diffusion of one metal in another sohd metal is in most cases an almost 
unilateral process. For example, the velocity of diffusion of gold in lead is very 
rapid hvit lead diffuses extremely slowly in gold. As the two allo;sing components 
become increasingly similar, for example, in passing from gold-lead, silver-lead, 
bismuth-lead, thalUum-lead, tin-lead to lead-lead, the one-sided nature of the 
process gradually disappears. Diffusion measurements make it possible to determine 
very low solubilities of one metal in another. The solubility of silver in lead at 
285°C was thus found 1o be 0.13 atomic per ceni . 

^ In the litcratvH-c silver is stated to be insoluble in solid lead. 



Originally publishod in Phijs. Z. 56, 790 (l<)2!)) 

14. THE APPLICATION OF RADIOACTIVE RECOIL 
IN DIFFUSION MEASUREMENTS 

G. Hevesy iuul \\'. Seith 
From lli(> Institulo of Physical Chemistry, Universil_\ of Froiburg 

A LAYER ol" thorium-B chloride placed on the surface of PblJlg show s a 
decrease in a-recoil yield after heating. The velocity of diffusion of Ihc 
thorium-B ion in lead chloride and thus the velocity of self-diffusion 
of the lead ions can be determined from this effect. This extraordinarily 
sensitive method by means of which diffusion constants down to 10 ^"^ 
cm2 day"^ can be determined permits the measurement of the velocity of 
diffusion in Pb( l, and Pblg in the vicinity of 100°C or at a higher 
temperature. 

Two different cases must be distinguished in diffusion in crystalline 
substances, heterogeneous diffusion and self-diffusion. The difference 
between these two cases exists also in other states of aggregation but is 
only slightly perceptible in the liquid and gaseous forms. During hetero- 
geneous diffusion in crystalline substances individual lattice components 
are replaced by foreign particles or the foreign ions (atoms) intrude into 
the interstices of the lattice. In self-diffusion the lattice components are 
replaced by identical particles, (.'onsiderable affinities between the 
diffusing and lattice-element substances often operate during heteroge- 
neous diffusion and we are confronted with a process which is a combi- 
nation of a chemical reaction, often proceeding with a significant decrease 
in entropy, and a true diffusion process. Self-diffusion produces merely a 
positional mixing of the lattice components without any practicable change 
of entropy. The phenomenon of self-diffusion is employed when informa- 
tion is required on the strength of binding of the individual ions (atoms) 
in the crystalline compound. In such measurement the method often used 
is to study the diffusion of an ion, e.g. a cation, in the crystalline compound 
whose cations are closely related to the diffusing one. For example, the 
diffusion rate of cuprous ions in silver salts may be measured or that 
of the cuprous ions in silver salts the cuprous and silver ions ])eing 
considered to be nearly identical from the standpoint of diffu- 
sion. The binding strength of silver and cuprous ions in various 
compounds can be determined to a fair approximation from such 
measurements. On the other hand i1 is not possible to determine the 



12H ADVENTURES IN RADIOISOTOPE RESEARCH 

binding strength of e.g. iodide in silver iodide, by similar measurements, 
and it must suffice to conclude from Tubandt's^ transport measurements 
and G. G. Schmidt's ionic emission experiments that the binding strength 
of the iodide ion in silver iodide is considerably greater than that of the 
silver ion. The indication of the last-mentioned result calls to mind another 
method for determining the velocity of self-diffusion, viz. by calculating 
from the electrolytic conductivity of the crystalline compound by using 
the theory propounded by Nernst for electrolytic solutions or by means 
of the Einstein diffusion equation. This method also, however, yields 
only the velocity of self-diffusion of the lightly bound ions, that is, silver 
and cuprous ions in silver and cuprous salts. 

The ideal of self-diffusion can be approached extraordinarily closely ))y 
allowing a radioactive ion to diffuse in the appropriate compound of 
an isotopic inactive ion, by applying the method of radioactive indicators, 
for example, by measuring the diffusion of ThB ions in lead chloride. 
Now the ions of the radioelements, except those of the thalium isotopes 
which are too short-lived (half-life always less than 5 min) to be considered, 
are multivalent. Multivalent ions, however, are always characterized 
by particularly strong binding^. From this it follows that the self- 
diffusion can be measured by the method sketched out above only 
with the aid of an extremely sensitive arrangement. The values thus 
obtained should indeed yield data on the binding strength of even this 
ion which has practically no share in the electrolytic conductivity and 
whose velocity of self-diffusion cannot therefore be calculated from 
conductivity data. With the usual measuring apparatus the procedure of 
Stefan is followed by placing several, frequently three, equally thick 
layers of the diffusion medium on a layer of the diffusing substance. 
The diffusion constant is inversely proportional to the square of the 
layer thickness. The smaller the diffusion constant to be measured the 
less will be the chosen layer thickness. If a velocity of diffusion (D) of, 
for example, 10"^ cm^ day~^ is to be measured then, for an experimental 
time of 1 day, it is necessary to choose a layer thickness of about 0.01 
mm. It is not practicable, however, to place three equally thick inactive 
layers on a 0.01 mm thick layer of radioactive lead chloride and to 
separate them again after diffusion for the purpose of radioactive anal- 
ysis. On the other hand the various radioactive methods yield opportuni- 
ties to attain such small layer thicknesses in another way. One of the 
authors with Obrutschewa^ has determined the velocity of self-diffu- 
sion of lead atoms, both in single crystals and in crystalline lead, by 



H\ TuBANDT, H. Reinhold and W. Jost, Z. pltys. Chcin. 29, G9 (1927). 
"Compare the transport results of Tubandt, Z. phys. Chem. 29, 69 (1927) ; 
s(>o also E. Friedrich, Z. Elektrochem. 32, 576 (1926). 

^G. Hevesy and A. Obrutschewa, Nature 115, 674 (1925). 



THE APPLICATION OF RADIOACTIVE RECOIL Ix\ DIFFUSION MEASIREMENJ .s 1 2i) 

collecting atoms of the lead istotope on a lead surface and counting the 
number of scintillations shown by the infinitely thin radioactive coating 
before and aft(>r diffusion in the heated metal. The decrease in the num- 
ber of scintillations is a measure of the velocity with which the radioactive 
lead atoms have passed, because of diffusion, outside the range of the 
a-rays and into the interior of the metal. The layer thickness here required 
toi- the diffusion calculation is the range of the a-rays in lead, which 
amounts to about 1/30 mm. Diffusion constants down to lO-s cm'^ day-i 
were measured with the help of this method. This sensitivity, however, 
was inadequate in the present study and we therefore used the radioactive 
recoil effect which can be expected to yield a considerable increase in 
sensitivity for detection of the diffusion. The range of a-recoil in lead 
amounts to only 3x10-^ mm and thus the application of the recoil 
method permits measurement of the extraordinary small diffusion 
constant of 10-^=^ cm^ day ^ The velocity of diffusion of the ions of the 
lead isotope ThB (half-life 10.6 hr) was measured in different compounds 
lead. The recoil yield thus the activity of ThC, was determined before 
and after diffusion. The radioactivity measurements were made only 
after the establishment of the equilibrium between ThB and ThC, since 
the a-rays responsible for the recoil effect are derived not from thorium-B 
but from its daughter product thorium-C (half-life 1 hr). 

EXPERIMENTAL METHOD 

The radioactive substance was condensed from the vapour phase on 
the pellet to be used for measuring the velocity of diffusion. The pellets 
were prepared by pressing very carefully purified lead chloride or iodide. 
The pressure used was 1800 kgm/cm^ and was applied for 1 min. 

The pellet was pressed on to the front of a 14 mm diameter brass 
cylinder and the two together were suspended in the apparatus (Fig. 1). 
This apparatus consists of two chambers A and B connected by means of 
a cock with a 2 cm bore. Each chamber can be separately evacuated and 
filled with purified nitrogen. The brass cylinder with the pellet is fastened 
l)y means of a silver wire to a screw, C, situated above the chamlx'r 
.4. A phosphorus pentoxide tube is first attached to the standard joint. 
8, and the whole apparatus is filled with nitrogen. 

The ThB chloride is condensed on to the lead chloride surface in t he 
following way : the active deposit from thorium is collected on a plati- 
num foil and the foil is then exposed to the action of chlorine gas. The 
foil is transferred into the vaporization apparatus, which is attached at 
S (see Fig. 1) while the pellet is situated in the chamber A. The vapor- 
ization chamber consists of a brass tube K, which can be cooled, into 
which the brass cylinder with the pellet just fits. The lower joint of 



9 Hevesy 



130 



ADVENTURES IX RADIOISOTOPE RESEARCH 



this tube is connected with a matching glass joint, which contains two 
brass rods for the power supply and across the ends of which the activa- 
ted platinum foil is fastened horizontally. The chamber B and the 
vaporization chamber are evacuated and filled with nitrogen before the 
pellet is lowered from A until it is near the top of the platinum foil G. 
At a nitrogen pressure of 1 mm the foil is brought to a white heat (about 
900°) for 1 sec and thus the vapour of ThB chloride is transferred on to 
the surface of the pellet. 




Pumpe 



Fig. 1. 



The recoil product, ThC", emits ^-rays whose strength constitutes 
an easily traceable measure of the recoil yield. The recoil product is 
collected as follows : After first waiting until radioactive equilibrium has 
been attained the pellet is placed in a tube attached at 8 above a copper 
foil charged to —220 V, the pellet being earthed, and the recoil atoms are 
collected on the copper foil. A decrease in the pressure to 2 cm aids the 
collection of the recoil product. Measurement of the /5-activity of the 
copper foil gives the recoil yield before the diffusion. 



THE APPLICATION OF KADIOACTIVK KECOIL IN DIFFUSION MEASUREMENTS I'.i J 

The pellet can b(> hiought up to the experimental temperature for a 
definite time by attaching at S a furnace containing a glass vessel with a 
standard joint. In the glass vessel is a hollow iron block (1.5 kgm) into 
which the pellet and the brass cylinder can be introduced such that the 
direct contact with the metal facilitates rapid equalizationof the temper- 
ature. The temperature is measured by Hoskins' method by means of 
a thermoelement of high thermoelectric power fixed in a side hole in th(^ 
iron block. This apparatus also is filled with nitrogen. 

The lead chloride was prepared from Kahlbaum purest lead chloride 
by repeated recrystallization from hydrochloric acid solution. It was 
dried by heating nearly to the melting point in a current of HCl. 

The lead iodide was obtained from an active acid solution of HI and 
Pb(N03)2, purified by decantation and dried over PgOg. Sublimation 
was avoided since Pbig prepared thus always contains traces of iodine. 

CALCULATION 

AVe are indebted to Professor R. Fiirth of Prague for the formulae 
used below. 

The calculating procedure is as follows : If the recoil activity before 
the experiment is equal to 1 and after the experiment to A, then 1—^ 
atoms of lead have diffused so far in to the pellet that their recoil products 
are no longer able to leave it. If all the recoil particles moved perpendi- 
cular to the surface, then all those issuing from lead atoms which had not 
diffused deeper than the range, a, of the recoil particles would be able 
to escape from the surface. The relationship between the number A and 
the diffusion constant D is therefore 



where Z is the time and x is the distance of the particle from the surface. 
By using the Gaussian error function 



(2) 




the equation 

A — an _ 

2]/DZ 
is obtained, whence D can be calculated 



A^yji-^] (3) 



132 ADVENTURES IN RADIOISOTOPE RESEARCH 

It must be home in mind that in the present instance the recoil par- 
ticles are expelled in all directions, such that the particle can reach the 
surface only when the distance x of iis starting point from the surface 
satisfies the condition 

x/at t^ cos a (4) 

where a is the angle between the normal to the surface and the ray. 
The ratio of the number of particles reaching the surface from a point 
C to the total number of particles issuing from that point in all directions 
is equal to the ratio of the surface of the spherical cap of height a~x to 
the surface of a sphere of radius a, whence it follows that 

2 7t a(a — x) , X 

^ ^^^ = 1 (5) 

2 7ia^ a 

To take account of this concept the integrand in (equation (1) must be 
multiplied by {l—x/a), and thus 



a 



A=.\-J 



]f{7ii)Z) 



1 e- •'"''^*^ • dx 1 6) 



a 



or, by substituting 
the result is 



I = a/2y{DZ) 



A = ^p{^)-^{}-e-''} (8) 



This equation is evaluated graphically and from the value of | thus 
obtained D is calculated by means of equation (7). 

Since the diffusion constant has been calculated in some cases from the 
decrease in the a-ionization it may be appropriate to discuss the calcula- 
tion for that method. The a-activity was measured by selecting a parallel 
])eam normal to the pellet surface such that equation (1) could be applied. 
The shutter had an air gap of 5.3 cm and thus only the a-rays of Th('", 
with a range of 8.4 cm, were able to penetrate. The conditions for the 
calculation were thus simplified. In measuring D by means of a-racliation 
it must l)e noted that the particles entering the electroscope do not all 
have the same ionizing effect since this is dependent on the path already 
travelled by a particle. A particle which has come from the interior of the 
pellet has less effect than one which has started from the surface. If the 
decrease in ionization due to a particle which has travelled a distance 
X in the Pbia pellet in relation to the effect of one which has started from 
the surface is represented by 

J = (fix) (9) 



THE APPLICATION' OK RADIOACTIVE KKCOILI.N DirjrSlQN MEASUREMENTS 133 

and if also the retarding efl'ect due to the air column of the shutter is 
equal 1o that of a Pbig layer witli a thickness b, then 

a—b 

J ]l{nDZ) ' 



In llie al)()\(' equal ion 

I = {a-h)!2]f{DZ) (11) 

and the expression is evaluated grapiiically. 

THE DIFFUSION OF LEAD IONS IN LEAD CHLORIDE 

If an at1(Mn])t is made to determine the diffusions of huid ions in lead 
chloritle witli ihe ordinary apparatus of Stefan, by pressing together a 
3 mm deep inactive lead chloride pellet and a 1 mm deep ThB-labelled 
lead chloride layer, no diffusion of the radiactive lead ions can be detected 
after 4 days at 480°C. It is not feasible to raise the experimental 
temperature since the high vapour pressure of lead chloride at the 
above-mentioned temperature (lO^i mm) already causes disturbance. 
The interference can indeed be partly restrained by carrying out the 
experiment in a pressure bomb under a nitrogen pressure cff 200 atm, 
but cannot be wholly eliminated. For the reasons mentioned it also seemed 
hopeless to prolong the duration of the experiment, possibly by replacing 
ThB by the long-lived RaD. For the same reason, the determination of 
the diffusion constant of lead chloride, in the vicinity of the melting point . 
by means of the decrease in a-radiation after heating ThB chloride col- 
lected on the surface of a PbCla pellet was also a failure. Even at 370( '° 
the amount of ThB which evaporates can be detected by radioactive me- 
thods. The diffusion constant of lead ions in lead chloride must therefore 
be measured at lower temperatures at which only the very sensitive recoil 
method can be considered. The results of the measurements obtained by 
this method are shown in Fig. 2 and Table l.The circles relate to a series 
of experiments in which the active deposit was treated with chlorine. 
The circles combined with strokes relate to experiments where ThB 
oxide or sulphide, instead of the chloride, was condensed on the PbClg 
pellet. Another series of results not quoted here yield Ihe same graphical 
pattern. 

The time of experiment was so chosen that the decrease in the recoil 
yield after diffusion amounted to about 50 per cent. This circumstance 
is most favourable both for carrjdng out the experiment and for calcula- 
tion. It is necessary to know the range of the recoil rays in lead chloride 
in order tobeable to ealeulate the diffusion constant from equation 8 on 



134 



ADVEXTTIRES IN RADIOISOTOPE RESEARCH 



page 130. This quantity is determined as follows : The retarding power 
of lead and of chlorine for a-radiation is known, whence the range of u- 
radiation in PbClg can be obtained. The ranges of a-radiation and recoil 
particles in air are known. On the assumption that the ratio of the 
ranges in air is equal to the ratio in lead chloride, the range of recoil 
particles in lead chloride calculated from the above data is 7.5 X 10-^ cm. 

-5 
'S 

-7 

-S 

-9 

-10 

-11 
logD 

~^^ 125° 150° IPS'* 200° 225° 250° 275° 300° t 

Fig. 2. Self-diffusion of Pb in PbClg and Pbl.^. a Strahlon = a-iadiation 
RuckstoBstrahlen = recoil radiation 





r 1 












.0^ 














X. 


^^ 










^A 
















:^^- 


w 




y^ 


Z' 






r^ 


y 


%<p 


J\ 


Y 








Jp 


y 




9^f^ 


V 










^ 




/ 


y 













in the discussion of the experiments with Pbig a method will be described 
which permits experimental testing of the correctness of the above 
value. In order to make sure that vaporization effects have not influ- 
enced the results experiments have also been performed at reduced 
pressure and these have yielded the same results as those at the ordinary 
pressure. 

Attempts to condense TliB oxide or sulphide instead of the chloride 
were made, in order to obtain information on the effect of a possible 
incomplete formation of ThB chloride on the experimental results. It is 
evident from Fig. 1 that the results were not essentially different, owing 
to the fact that the lead ion surrounded by many chlorine ions soon loses 
its oxygen partner. The treatment of the PbOlg pellet with chlorine or 
HCl. after condensing the active material, is also without effect on the 
result. 



THE APPLICATION OF RADIOACTIVE RECOIL IN DIFFUSION MEASUREMENTS 135 



Table 1. — Self-Diffusion of Pb Ions in PbCl, 



t 


i/r 


D 


logZJ 


Roraarks 


166 


0.002277 


1.47 


10-12 


— 11.83 




180 


2207 


4.20 




— 11.38 




180 


2207 


4.44 




— 11.35 




183 


2193 


6.60 




— 11.18 




198 


2123 


2.38 


10-" 


— 10.62 




201 


2108 


2.72 




— 10.57 




210 


2070 


5.79 




— 10.24 




211 


206S 


4.69 




— 10.33 


Sulphide distilled 


216 


2045 


9.68 




— 10.01 


Oxide distilled 


217 


2040 


9.93 




— 10.00 




220 


2028 


1.28 


10 10 


— 9.89 




223 


2018 


1.22 




— 9.91 




225 


2008 


1.63 




— 9.79 


Oxide distilled 


225 


2008 


1.85 




— 9.73 




235 


1969 


3.44 




— 9.46 




249 


1916 


9.00 




— 9.05 




268 


1847 


2.60 


10-9 


— 8.58 




270 


1841 


3.16 




— 8.50 





The curve of Fig. 1 can be represented by the equation 



D = 1.060x107 p-38.i2o/«r 



This yields 38,120 cal for the molecular heat of relaxation of lead ions 
and the value 1.06 x 10-^ for the constant ^. The heat of relaxation of the 
chloride ions in lead chloride is found to be 10,960 cal, from the conduct- 
ance of PbCla- The large difference between the heats of relaxation 
immediately renders intelligible the result of Tubandt, according to 
w hich practically all the mobility in lead chloride is due to the chloride 
ions. The lead ions require a much greater energy content than the 
chloride ions to enable them to take part in place exchange processes. 
The transport number of lead ions in lead chloride is found to be 10-^ 
at 270"C. 

The investigation of self-diffusion in single crystalls of lead chloride, 
in which the present experience suggests a smaller diffusion, will be 
discussed later. 



130 ADVEXXrilES IN E.\DIOISOTOPE RESEARCH 

THE DIFFUSION OF LEAD IONS IN LEAD IODIDE 

Lead iodide has a smaller electrolytic conductance llian lead chloride ; 
yet a relatively high diffusion velocity of lead ions in lead iodide would 
be expected, in spite of the low conductance, since Tubandt found a 
high value (0.67) for the transport num])er of the lead ion in Pblg. In 
agreement with this expectation, it is evident from Table 2 that the 
diffusion constant can be measured even a few degrees above 100°(J. 
It was always necessary to use annealed pellets in order to obtain repro- 
ducible A^alues. 

We find the molecular heat of relaxation to have a value of 30,000 
cal and the constant A to amount to 3.43 10^. 



'lABLr: 2. - 


- Self-Diffusion of Le.\d 


Ions ix Pbl 




(Recoil 


Method) 




/ 


1/7- 


/' 


loj D 


114 


0.002585 


6.31 • 10-12 


— 11.20 


122 


2532 


9.59 


--1I.02 


124 


2518 


1.47 • 10-11 


— 10.83 


137 


2440 


4.23 


— 10.37 


147 


2382 


1.17 • 10^10 


— 9.93 


165 


2283 


6.35 


— 9.20 



The diil'usion constant can be represented by the formula 

/) z= 3.43 X 10^ e 30.000/flr 

Since there is an appreciable mobility of the lead ion in Pbig even a1 
temperatures which are very far removed from the melting point it was 
also possil)le to apply the decrease of ionization after diffusion, due to 
the a-particles, for measuring the diffusion constant. The results are 
shown in Table 3 and Fig. 2. The diffusion constant can be represented 
))y table following equation : 

Z) = 9.11 X 10^ e-3oi40/7?r 

The heal of relaxation, which amounts to 30,140 cal/mole, does not 
difler appreciably from the value yielded l)y the recoil experiments 
(30,000 cal). This agreement is also expressed in the parallel courses of 
the curves in Fig. 2. The fact that they do not quite coincide is probably 
due to some uncertainty attaching to the value of the recoil range, as 
already mentioned above. The two straight lines could be superposed by 
assuming the range of the recoil particles in lead iodide to be 0.11 // 
instead of 0.075 //. 



THE APPLICATION OF RADIOACTIVE RECOTI.TX DIFFUSIOX MEASIREMEXTS 137 



Tabic 3. — Self-Diffttsion of Lead Ions in Phi, 



(q-Particle AIethou) 



t 


•/•/• 


" 1 


log 1) 


255 


0.001805 


3.63 • 10-^ 


—6.44 


260 


ISTC) 


5.30 


-6.2S 


301 


1749 


3.42 ■ 10" 


— 5.47 


302 


1739 


4.26 


—5.37 


315 


1701 


6.70 


—5.17 



Th(» (liilusion constant of lead ions in Pbl, can also he calculated ironi 
the electrolytic conductance of this compound and the transport number. 
At 390°C, for example, the calculated diffusion constant is 0.9x10 •> 
cm2 day~i while the recoil measurement and a -ray measurement yield 
0.9 X 10"^ and 2.2 x 10-^, respectively. The behaviour at low tempera- 
tures, where the mobihty of the iodide ions controls the conductanee, 
is discussed in the subsequent paper. 



Summary 



The veloeitA- of diffusion of leail ions in lead chloride and iodide has been 
measured by making use of radioactive recoil. The values obtained are DpbOij = 
= 1.06 x 10"e-38'i2o/iJ?'and£)pi,i, = 3.43 X 10* e-^^'Ooo/^?' . The diffusion velocity 
values for the lead ion in lead iodide were confirmed by other methods. The high 
value for the heat of relaxation of lead ions in lead chloride (3S,120 cal/mole) 
cixplains Tubandt's result, viz. that in lead chloride the chloride ions whose 
heat of relaxation amounts only to 11,180 cal are practically the only mobile 
ions. The transport number of the lead ions in lead chloride at 270" is cal- 
culated to be 10~5. 

The velocity of diffusion of lead ions in lead iodide calculated from the electro- 
lytic condu<*tance and from the transport numbers at 290°C as determined by 
TuBANDT, is in good agreement with our experimental value. 



138 ADVEXTURES IX RADIOISOTOPE RESEARCH 



Comment on papers 10 — 14 

When faced with tlie task of calculating the diffusion rate of gaseous oxygen 
in gaseous nitrogen, to facilitate the calculation Maxw^ell made the assumption 
that the molecules of oxygen and nitrogen have the same radius and same mass ; 
he thus arrived at the notion of self -diffusion. The introduction of the labelling 
principle made it possible to measure a diffusion rate close to self- diffusion. 

In connexion with the discussion of the interchange between lead atoms of a 
lead foil and the surrounding lead ions the problem of the diffusion rate of solid 
lead in lead was first raised in 1915 (paper 8). The first experiments in this field 
described in paper 10 were, however, carried out a three years later only. Prior to 
this investigation Groh and the wTiter measured the rate of diffusion of labelled 
molten lead in non-labelled lead. Self-diffusion in liquids cannot be expected to 
lead to results which cannot more or less be foreseen. The rate of diffusion of 
molten lead in molten lead does not differ much from the diffusion rate of cadmiuiu 
or thallium in molten lead. In contrast, the rate of seh-diffusion in solid metals 
cannot be foreseen. The diffusion of a solid metal, even a closely related one, 
in another metal produces changes in the crystalline state which may strongly 
facilitate penetration. We found that the atoms of solid labelled lead diffused 
into solid lead about 200 times slower than thallium atoms and about 10,000 
times slower than gold atoms diffuse into solid lead. In the first investigation 
on the diffusion in solids described in paper 9 labelled lead was soldered on a 
non-radioactive lead rod. After keeping this system at 280°C for up to 400 days 
shces from the rod close to the place of soldering were prepared and their radio- 
activity compared. The figures obtained permitted the calculation of the upper 
limit of the diffusion rate of lead in lead. In a later investigation (paper 11) 
carried out with Mrs. Obrutsheva, wife of the weU-known Russian physical 
chemist Frumkin, we increased the sensitivity of the method b;^- pressing in 
vacuo a non-radioactive lead foil on one labelled with thorium B. The thickness 
of the inactive foil was chosen slightly greater than the range of a-particles to 
be measured ; therefore no scintillations originating from the radioactive lead 
could be observed when investigating the inactive foil. But, on heating the aggre- 
gate of the foils, a diffusion of the active lead into the inactive one took place 
and the a-particles emitted by the succession products of ThB (ThB emits no 
rx-rays, but comes rapidly into exchange equilibrium with a-particles emitting 
desintegration products) produce scintillations on the observing screen. In further 
investigations with Seith we replaced the counting of scintillations by ionization 
measurements. The range of a-particles emitted by the disintegration product 
of ThB in lead amounts to 3 x 10" ^ cm. A replacement within this thickness 
of some of the ThB atoms by non-radioactive lead atoms due to an interchange 
process leads to a decrease in the ionization measured. The range of the recoil 
particles emitted by the ThC, the disintegration product of ThB, is stiU appreci- 
ably (almost 1000 times) shorter than that of the a-particles. The measuring of the 
decrease of the recoil yield with time of a with ThB covered surface perinits to 
determine as low a diffusion rate as 10~i3 cm^day^ By making use of this me- 
thod self-diffusion in solid lead taking place at 106°C or at a higher temperature 
Avas measured (paper 12). From the change of the rate of self- diffusion with 
temperature the value prevailing closely to the melting point was calculated 



13<) 



iiiul compared with the rate observed after melting took phur. The ratio wor- 
ked out to be 10,0()(). 

The measurement of the rate of self-diffusion of lead ions in a solid lead salt 
permitted the determination of the transference number of the ions of solid lead 
lialogenides (paper 14). In his very beautiful investigation Tubandt found that 
while both ions have a large part in the conduction of electricity through the 
solid lead iodide, alone the movement of chloride ions is responsible for the 
jnissage of an electric current through solid lead chloride. The ionic mobility of 
Ph-' in PbCl, calculated from its self-diffusion rate determined by making use 

ol the very S(!nsiti\e lecoil method indicates that for about - pait of the 

(>iectrical curient passing through solid lead chloride the movement of lead ions 
is responsible. 



References 

I. Groh and G. Hevesv (1920) Ann. Phys. 63, 85. 
G. Hevesy and W. Seith (1929) Z. Phys. 57, 869. 
G. Tubandt, H. Reinhold and \V. Jost (1928) Z. Anorg. Chem. 177, 254. 



Originally published in Nnturc 125, 744 (1930) 

15. SEARCH FOR AN INACTIVE ISOTOPE OF 
THE ELEMENT 84 (POLONIUM) 

G. Hevesy and A. Guenther 
From the Institute of Physical Chemistiy, University- of P'reiburg 

The elements 81 (thallium), 82 (lead), and 83 (bismuth) have both 
radioactive and inactive isotopes, whereas the elements 84 — 92 are only 
known in an active form. Several attempts have been made to find 
inactive isotopes of the latter elements. Aston, using his mass spectro- 
graph, tried to discover a stable isotope of radon in the atmosphere, 
and Hahx made extensive researches to find an inactive isotope of 
radium. All these attempts failed. 

We have recently tried to extend the series of inactive elements by 
searching for an inactive isotope of the element 84 (polonium), which 
follows bismuth. Through the work of the discoverer of this element. 
Mme. Curie, and her co-workers, as avcH as of Marckwald and of 
many others, the chemical properties of polonium were found to be 
intermediate between those of bismuth and tellurium. Hence it is obvious 
that if a stable isotope exists, it must be associated in nature with 
tellurium or bismuth. 

We looked for the elements 84, therefore, in the following tellurium and 
bismuth minerals : Hessite, calaverite, nagyagite, tetradymite, and 
bismuth glance as well as native bismuth. The minerals were dissolved, 
and a known amount of polonium added as radioactive indicator. On 
removal of the polonium from the solution, it was to be assumed that 
any isotope present in the solution would accompany the active polo- 
nium. By special methods devised for the purpose, it was possible to 
legain the added polonium electrolytically on molybdenum electrodes, 
the deposit weighing only about 1/10 mgm. X-ray investigations, carried 
out by the secondary ray method to avoid the possible volatilisation oi" 
the substance under the action of the cathode rays, have shown that the 
deposit cannot contain more than 1/2 per mille of the element looked 
for. The X-ray line searched for was polonium L^^ , the wave-length of 
which was calculated from Moseley's law to be 1111 X. U. All the lines 
on the plate could be identified as belonging to lead, bismuth, silver, 
mercury, or tungsten. As we started with about 400 grams of each of the 
minerals mentioned, 1 gm of each mineral cannot contain more than 10-" 



SEARCH FOK AN INACTIVE ISOTOPE OF THE ELEMENT t<4 (POLOMI .M) 141 

om. of the clement in question. 'I'his negative lesuH is in agreement 
with generalisations arrived at by Dr. A. S. Russell. 

There is very little hope of finding an inactive polonium isotope, 
or in general of extending beyond bismuth (83) the series of stal)le 
elements. 



142 ADVENTURES IN RADIOISOTOPE RESEARCH 



Comment on paper 15 

The search for unknown elements is much facilitated by adding a radioacti\(' 
isotope of the desired element to the solution of minerals in which the element 
is most hkely to be present. In paper 15 the conclusion was reached that a heavier 
stable element than bismuth is unlikely to be found. This conclusion is supported 
by later experience. 

In an earlier investigation carried out in 1926 in Copenhagen an unsuccessful 
search was made for a stable isotope of the element 87 by trying to detect a-rays 
emitted by MsThj or )S-rays by radon. Both processes should lead to the formation 
of the element 87. A radioactive isotope of this element was later discovered 
by Perey and Lecoin. A more detailed presentation of the results stated in 
paper 15 is to be found in Z. f. anorg. Chem. vol. 194. 



References 

G. Hevesy (1926) Kgl. Danske Vid. Selsk. Mathem.-fysiske Medd. 7, 11. 

M. Perey and I- Lecoin (1939) Nature 144, 326. 

G. Hevesy and A. Guenther Z. /. anorg. Chem. 194. 162 (1930). 



Originally published in C. R. Acad. Sci., Paris 178, 1324 (1924) 

16. RADIOCHEMICAL METHOD OF STUDYING THE 
CIRCULATION OF BISMUTH IN THE BODY 

I. A. Christiansen, G. Hevesy and S. Lomholt 

Fi-om the Institute of Theoretical Physics, University of Copenhagen 

During recent years l)ismuth has acquired increasing importance in 
the treatment of syphilis^i^ In order to study the conditions of absorption, 
distribution in the body and elimination, we have used a radiochemical 
method which was first proposed by Hevesy and by Paneth^^) 

The principle of the method is as follows: The medicament is prepared 
from a mixture of a bismuth salt solution and a solution containing a 
radioisotope, radium-P], of this element. It is well known that a final 
measurement of the quantity of radioisotope present in the sample 
suffices for calculating the quantity of inactive bismuth. 

Radium-E was extracted with a hydrochloric acid solution from 
radium-D, which had been produced by disintegration of the emanation 
from a quantity of radium corresponding to 2—4 c. The experiments 
were performed on rabbits. From time to time small quantities of an oil 
suspension of the medicament were injected intramuscularly. The rabbits 
were killed after about 15 days. 

The following samples were examined: (1) the places of injection; 
(2) the most important organs; (3) the daily amount of urine; (4) the 
daily amount of faeces; (5) small known amounts of the suspension used 
in the experiment. 

All the organic tissues were prepared for analysis by charring with 
small quantities of fuming nitric acid; the ash was dissolved in dilute 
nitric acid and the acid was evaporated of in a petri dish; the radio- 
activity of the small quantity of salts remaining at the bottom of the 
capsule was finally determined electroscopically by measuring the /5-rays 
of the radium-E. The a-rays from the polonium present in the residue 
were absorbed by means of an aluminium foil about 0.05 mm thick. 
Control experiments have shown that the maximum error of the various 
experiments performed by this method is about 10 per cent. 

1 Sazerac and Levaditi, C. R. Acad. Sci., Paris 172, 1391 (1921); Ibid. 173, 

338 (1921). 
- See Aston, The Isotopes, London (1923); F. Paneth, Z. atigew. Chem. 35, 549 

(1922); G. Hevesy, Biochem. J. 17, 441 (1923). 



144 



AI)Vf:\TrJ!KS IX IJADIOSIOTOPB RESEAllCH 



Nine rabbits were used in these experiments. Quinine Insmuthiodide^^' 
was used in live cases and bismuth hydroxide in the remaining four. 
The results obtained were in reasonable agreement. We shall limit the 
results presented here to those from one of the experiments with quinine 
bismuth iodide. These results are summarized in Fig. 1. The heights 
of the vertical columns represent the quantities of bismuth injected 
daily; the shaded parts of the columns represent the quantities found 



0,5 
0.4 
0,3 
0,2 
0.1 
0,0 






8> 

c 



a> 

c 
■o 



En 
o o 
O o 



mq 

2,65 
2.50 



2.00 



,50 



.00 



0,50 



Faeces 



0,0 .Urine 




/2 2/2 '/fe "/Z Vz 6^ 7/2 8/2 9/2 lO/j U/a 



Fig. 1 

Di Iribution of bismuth in iho i-aV)bit 



at the corresponding points of injection. The upper black columns 
represent the quantities found in the daily faeces and the lower black 
columns the quantities found in the urine. The black rectangles 
placed in the upper part of the diagram give the contents of the various 
organs. 

The general results of all the experiments can be summarized as fol- 
lows : ^1* bismuth is eliminated chiefly in the urine; the quantity of bis- 
muth found in it is double the amount passed in the faecal matter; 
it increases during the period of treatment in the urine but this is not 
so clearly demonstrated in the faecal matter; (2) the heart and lungs 
contain only a small amount of bismuth; the liver contains quite a small 
(juantity and the kidneys a fair amount, generally mor(> than d()ul)le 
lhe amount in the liver; only a very small quantity of bismuth has been 
lound in 50 cm'^ of l)lo()d. 



METHOD OF STTDVINT; TMi: ('IlM'tr.VTTOX OF lUSMTTir T\ TTTK IM)I)V 145 

The results obtained show that bisinutli should only bo usod with 
great care, because of its quite slow and irregular distribution leading 
possibly to a danger of poisoning. Its resemblance with mercury^^^ from 
ihe aspect of circulation. Precautions are required in the simultaneous 
use of the two metals, Avhich cause accunuilation of Ihe toxic effect. 



^ See SvEND LoMHOLT, BHt. J. Dermatol. (1921); Arch. Derniatolog. n. Si/philis 
126, 154 (1918). 



] Hovcsy 



Originally published in C. B. Acad. Sci., PaHf> 179, 291 (1924). 

17. RADIOCHEMICAL METHOD OF STUDYING THE 
CIRCULATION OF LEAD IN THE BODY 

I. A. Christiansen, G. Hevesy and Sv. Lomholt 

Fi-om the Institute of Theoretical Physics, University of Copenhagen 

At the meeting of the Academy on 7 April 1924 we presented some work 
performed by a radiochemical method on the circulation of bismuth in 
ihe body. Since then we have used the same method for some experi- 
ments on the circulation of lead, the first results of which are presented 
here. 

In using bismuth in medicine it is of very great importance to know 
the rate at which the bismuth injected into the body is distributed and 
eliminated. This question can be resolved by using a substance with a 
quite short half-life (5 days), such as radium-E. It is quite different in 
the case of lead, since all the medical interest in this element centres 
around the chronic poisoning which is caused gradually by the absorp- 
tion of small quantities of lead during a long period of time. This is the 
reason why we have used radium-D (half-life 20 years) in our experi- 
ments, whereas Hevesy, in his experiments on the distribution of lead 
in plants^i\ obtained satisfactory results with another isotope of lead. 
thorium-B, having a half-life of only 11 hr. 

Since radium-D emits only soft /3-rays, which are difficult to measure 
with the electroscope, we have counted the ^-radiation of radium-E in 
equilibrium with the radium D and, consequently, have measured the 
various products of analysis only at the end of several weeks. 

The experiments were performed on rabbits and guinea pigs. The 
method described previously has been modified slightly: Instead of 
evaporating the solution of the organic matter, which is decomposed 
by means of nitric acid (or potassium permanganate in acid solution), 
it is diluted, treatcfl with 100 mgm inactive lead nitrate, and lead sulphide 
is precipitated. After filtering at the pump on a plane filter, the dried 
filter is placed in a petri dish and the activity of the deposit is finally 
determined. 

One example^ only is given here from our experiments. The lead hydrox- 
ide, mixed with olive oil and a little carbon black, contained a quantity 

1. G. Heve.sy, Biochem. J. 17, 441 (1923). 



METHOD OF STITDYINU THK OIKCUl.ATION OK LKAD IX THE BODY 



14- 



oi radium-D derived irom the disintegration of an amount of emanation 
corresponding to Yo — ^ ('• The results are shown in Fig. 1. 

The heights of the vertical columns represent the quantities of lead 
injected daily; the shaded parts of these columns indicate the deposits 
of corresponding injections. The upper black columns represent llio 



o 

I 



c 

3 



> 
ZJ 



10 

>. 
a> 

c 



in 

o 

«> 
o 



o 

c 






o 

4) *- Q> 
r c o 



O O 

O o 

ID i3 




% 18 19 20 21 22 23 24 25 26 27 2f 



Fig. ] 
Distribution of lead in the rabbit 



quantities found in tlie daily faeces and the lower black columns show 
the quantities present in the urine. The black rectangles in the uppei- 
part of the diagram give the contents of the various organs. 

It will be evident that there is quite a substantial difference between 
the results for bismuth and for lead; the amounts of lead stored in tlie 
liver and eliminated in the faeces are larger, at the expense of the amounts 
found in the kidneys and urine which are the major participants in the 
case of bismuth. 

We have recovered 90 per cent of the amount of lead injected. 



10* 



148 ADVENTUftES IX JtAUIOISOTOrE RESEARCH 



Comment on papkrs 1(3, 17 

Ijst the early twenties dermatologists became much interested in the therapeutic 
application of bismuth compounds. This induced us, together with Christiansen 
and LoMHOLT (the latter being a dermatologist) to investigate the distribution 
of administered bismuth in the rabbit. We applied in our study RaE-labelled 
bismuth. This work was then extended to the study of the distribution of labelled 
lead. The first -mentioned investigation was the first application (1924) of radio- 
active tracers in animal physiology, shortly following their first application in 
plant physiology. 

While participating at the Liverpool meeting of the British Association for 
Advancement of Science, the writer learned that the gynaecologist Blair Bell 
obtained good results by applying lead salts in cancer therapy. This induced 
us (Hevesy and Wagner, 1930) to compare the distribution of lead between 
normal and cancerous tissue, applying labelled lead. The great Freiburg patholo- 
gist AscHorr on my request delegated a Japanese collaborator of his to help us 
in this work, and later Schoenheimer to assist the latter. This was the first 
oxperifcnce of schoenheimer in the field to which he later made, jointly with 
his eminent colleague Rittenberg, a very great number of unsurpassed classical 
contributions. 



References 

U. Hevesy and O. H. Wagner (1930) Arch. Exp. Pathol, and Pharmacol. 
149,336. 

R. Schoenheimer (1942) The Dynamic State Of Body Constituents-. Cambridge, 
j\lass. 



Originally publisliod in Xatuic 13, 754 (lO^f)). 

18. RADIOACTIVE INDICATORS IN THE STUDY 
OF PHOSPHORUS METABOLISM IN RATS 

O. Chievitz and G. Hevesy 
From the institute of Theoretical Physi(!S, University of Copenhagen 

Receis'T progress in the production of radioactive isotopes by neutron 
Ijombardment makes the radioactive isotope of phosphorus ?|P easily 
accessible. This isotope, which has a half-life value of 15 days, can be 
utilised as an indicator of inactive phosphorus in the same way that 
1 he radioactive isotopes of lead, bismuth and so on wen^ formerly used 



4- 






E 3H 

C 



3 
O 



Q. 



a I - 



O 

c 



+ 



o 

+ 



o 



o o 



o 






-| ' 1 ' — 

II 15 

Number of Days 



1^ 
19 



23 



27 



Fig. 1 . On the first day 7.4 per cent of phosphorus was found in the 
faeces and 5 per cent in the urine 



as indicators of these elements. If, for example, we add active ifP 
to 1 mgm of inactive phosphorus in such quantity that the Geiger 
counter registers 1000 impulses per minute, carry out with the phos- 
phorus activated in this way any sort of chemical or biological reaction 
and then find that the product obtained gives 1 impulse per minute, 
we may conclude that 1/1000 mgm of the phosphorus originally intro- 
duced is present in the product investigated. 



150 



ADVENTURES IN RADIOISOTOPE RESEARCH 



Rats were fed with a few milligrams of sodium phosphate containing 
J5P as indicator. The radioactive phosphorus present in the urine and 
faeces was then investigated for a period of a month. The result is shown 
in Fig. 1, which shows the percentage of the 2 mgm of phosphorus 
taken, found daily in the excrements. The rat was killed, and, after 
ignition, the phosphorus content of the different organs was investiga- 
ted. The result of an experiment in which the rat was killed 22 days 
after being fed on active phosphorus is seen in the first column in Table 1 . 
The largest part of the phosphorus taken is present in the bones, and 
the smallest in the kidneys. When, however, we take into account the 
very different weights of the different organs and calculate the phos- 
phorus content of the latter per gram after drying, we obtain a very 
different picture, as seen from the second column in Table 1. The spleen, 
kidneys, and the brain are found to contain per gram most of the active 
phosphorus. During one of the experiments, the rat produced six off- 
spring on the seventh day, of which five were eaten by the mother ; 
this caused a large increase in the active phosphorus content of the 
excreta in the following three days. The presence of 2 per cent of the 
2 mgm active phosphorus taken by the mother was revealed by the 
analysis of the remaining offspring. 



Table 1. 



Distribution of the active phospho- 
rus IN the rat 





Per cent 


Per cent 


Urine 

Faece.s 


26.3 

31.8 

0.5 

0.2 

1.7 

0.4 

24.8 

17.4 


per gm 


Brain and Medulla .... 
Spleen and Kidney.s . . . 
Liiver 


14.7 
18.2 
13.9 


Blood 

Skeleton 


1.8 

2.8 


Muscles and fat 


7.4 



The active phosphorus content of the urine and faeces shows great 
fluctuations during the first few days after the intake of the preparation. 
Later, it becomes fairly constant; and we have obviously to deal with 
the excretion of phosphorus which has already been deposited for a 
while in the skeleton, the muscles, or other organs, and wdiich has been 
displaced again. From our experiments, it follows that the average 
t ime which a phosphorus atom thus spends in the organism of a nor- 
mally fed rat is about two months. This is also supported by the fact 
that rats killed about a month after the intake of phosphorus contain 
only about half the active phosphorus found in those killed after a weeks 
time. This result strongly supports the view that the formation of tiic 



VTIOSVHOKI'S METABOLISM TX KATS 151 

bones is a dynamic process, the bone continuously taking up phosphorus 
atoms which are partly or wholly lost again, and are replaced by other 
phosphorus atoms. In the case of an adult rat, about 30 per cent of 
the phosphorus atoms deposited in the skeleton were roniOA(>d in Ihe 
course of twenty days. 

In another set of experiments we investigated the different parts 
of the skeleton. No conspicuous differences in the active phosphorus 
content could be found, with the exception of the teeth. The front teeth, 
which grow rapidly in rats, contained a larger part of the 2 mgm phos- 
])horus taken than the average of the whole skeleton, the ratio being 
about 10 : 1 in the case of adult and 6 : 1 in that of half-adult rats, 
whereas the molar teeth took up less than the average per gram of the 
skeleton, the ratio being 1 : 2 in the most extreme case. A detailed 
account of these and further results will be published elsewhere. 



Originally publishod in Kgl. Danske V ideu-shahevnes Selskah. Biologiske Meddeleher 

13, '.) (1037) 

19. STUDIES ON THE METABOLISM OF PHOSPHORUS 

IN ANIMALS 

O. Chievitz and Ci. Hevesy 
From the Instituto of Theoretical Physics, University of Copenhagen 

In a recent letter to Nature^ we communicated the results of some 
experiments on the metabolism of phosphorus using a radioactive 
phosphorus isotope as indicator. What follows is a more detailed descrip- 
tion of some of our experiments, carried out chiefly on rats but partly 
also on human subjects. 



PRINCIPLE OF THE METHOD USED 

Disregarding hydrogen, the only element which is ever met with in 
a nuclear state (as a proton) in chemical reactions, isotopes do not 
separate to a measurable extent during chemical or biochemical processes. 
It follows from this inseparability that Avhen a known amount of radio- 
active phosphorus is added to, for example, 1 mgm of phosphorus 
the presence of the former will always indicate the presence of the latter, 
we can thus distinguish for example between the phosphorus atoms 
taken in with the food (to which we add some radioactive phosphorus) 
and those already present in the system. The use of isotopic indicators 
is not dependent on an absolute inseparability of isotopes by chemical 
methods. We know indeed that minute separations almost always 
occur. It is sufficient that, within the analytical accuracy claimed, no 
separation takes place. 

Phosphorus has only one stable isotope ^^P but we can prepare un- 
stable radioactive isotopes of phosphorus having atomic weight of 
150 and 32; the latter has a half-life of about a fortnight and is \ery 
suitable for use as an indicator. It was used by us in many experiments 
of different kinds. 



10. Chievitz and G. Hevehy, Nature 136, 754 (1935). 



STUDIES ON THE METABOLISM OK I'lIOSl'HOKlS IN ANIMALS l.lli 

PREPARATION OF RADIOACTIVE PHOSPHORUS 

Radioactive ])lu)spli()rus ffP can ])v prepared from chlorine or IVom 
sulphur undei- ihe action of last iKHitrons, or from ordinary phosphorus 
under the action of slow neutrons; the nuclear reactions are: 

?fCl +ln = ?iP + file 

IIP +5n = ?fP 

Using neutrons liberated from mixtures of radium and beryllium. 
3^P can be prepared most conveniently from sulphur. We found it advis- 
able to use carbon disulphide instead of the elementary sulphur used 
by Fermi and his colleagues in their original experiments. About 10 
litres of carbon disulphide were exposed to neutrons from radium- 
beryllium mixtures and a fortnight later the carbon disulphide was 
distilled off. The residue contained the radioactive phosphorus formed, 
along with some of the decomposition products of carbon disulphide. 
The residue was oxidized and the phosphoric acid obtained converted 
into the phosphate compound wanted. We used chiefly sodium radio- 
phosphate in our experiments. The weight of the radiophosphorus 
produced is extremely minute; using a source containing 100 mgm 
of radium, less than 10" ^^ gm of radiophosphorus is obtained. By adding 
a suitable quantity of sodium phosphate to the sodium radiophosphate- 
solution we obtain the "radioactive" ("labelled") sodium phosphate 
desired. 

To concentrate the radiophosphorus ol)tained by neutron bombard- 
ment of carbon disulphide other methods besides that outlined above 
were used. A very convenient way to prepare nearly pure radiophos- 
phorus is the following. Under the action of the radiation some decompo- 
sition of the carbon disulphide takes place and a partly orange-coloured 
precipitate is formed which settles on the glass walls. This slight preci- 
pitate contains a large part of the radioactive phosphorus formed. 
The precipitate is possibly identical with the red sulphur described 
by Magnus as far back as 1954, which was found to consist of a mixture 
of sulphur and organic sulphur compounds. We are engaged on the 
investigation of this precipitate. 

In a third method of preparation the phosphorus formed was removed 
from the carbon disulphide solution by shaking the lattei' with diluted 
(20 : 1) nitric acid. 



] 54 ADVEXTURES IN RADIOISOTOPE RESEARCH 

DETERMINATION OF THE RADIOACTIVE SODIUM PHOSPHATE 

The radioactivity of the samples of blood, bones, etc. to be analysed 
is in most cases too feeble to be measured even by means of a very 
sensitive electroscope. Geiger-Mijller counters, much more sensitive 
instruments, are therefore utilised for measuring purposes. We use for 
the most part tubes having an available surface of about 1.5 cm^. The 
sample to be measured must accordingly be spread over about the same 
area. The j^-rays emitted by the radio phosphorus are fairly penetrating 
and are not much weakened when an aluminium dish of 1.5 cm^ 
surface is filled to a depth of a few millimeters with a bone sample 
weighing 100 mgm. We want to know what percentage of the radio- 
active phosphorus taken is to be found after a certain time in, for exam- 
])le, the bones. The procedure is as follows. We take a solution of active 
sodium phosphate, use 99 per cent of it for feeding the animal and keep 
1 per cent as a "standard". We kill the animal, separate a bone sample, 
ignite it, and measure its activity. Should the latter be, for instance, 
half as large as that of the standard which is measured simultaneously, 
then we can conclude that 0.99x0.5 per cent of the active phosphorus 
atoms eaten are actually present in the bone sample investigated. 
Although the jS-radiation from radioactive phosphorus atoms is not 
rauch weakened in penetrating through 100 mgm of bone ash, 
we can entirely eliminate the possible error due to this absorption by 
adding 100 mgm of calcium phosphate to the standard solution; 
this has the same absorbing power as the bone sample. It is advisable 
to make the standard as similar to the sample to be measured as possible. 
In dealing with urine, faeces, muscles, liver etc. we first destroyed the 
organic matter by one of the usual methods; in several cases, however, 
these were replaced by treatment with fuming nitric acid. Then calcium 
phosphate and calcium oxide were added if necessary to make the sample 
more similar in its composition to our standard preparation and finally 
the sample was ignited. 

To demonstrate the utility of the isotopic indicator method we 
will first consider the problem of the origin of the phosphorus in 
the faeces. 



ORIGIN OF THE PHOSPHORUS IN THE FAECES 

Chemical analysis enables us to determine the phosphorus content 
of the excreta but not to decide to what extent the phosphorus found 
in the faeces is undigested material and what fraction of it is phosphorus 
having its origin in the organism. The investigations described in this 
paper have revealed that a fairly rapid interchange takes place between 



STUDIES OX THE METABOLISM OF PHOSPHORUS IN ANIMALS 



155 



llio phosphorus present in the different bodily organs and that present 
in the blood. A part of the latter finds its way, when the digestive fluids 
are formed, into llie intestinal tract and is thus added to the faeces. 
The following experiment permits us to distinguish between food phos- 
phorus and that originating from the blood. We add a known amount 
of radioactive phosphorus to the diet and determine what percentage 
of the latter is to be found in the faeces. In a separate experiment we 
inject a known amount of radioactive phosphorus (sodium phosphate) 
into the blood and determine what part of this phosphorus appears 
in the faeces. The combination of the two results enables us to deter- 
mine what part of the phosphorus found in the faeces is due to incomplete 
digestion of the food eaten. 

In Table 1 the amount of radioactive phosphorus eliminated through 
the kidneys and the gut is given for the case of a patient fed on a normal 
hospital diet to which 0.5 mgm of labelled sodium phosphate was 
added. Within 5 days 21.7 per cent of the phosphorus was eliminated 



Table 1. — Radioactive Phosphorx's givex to 
HriiAN- Si'B.TECT Per Os 









Percentage of 


original 


Xuml 


er of days after 
taking P 


Diuresis 
in gm 




radioactive V 




in 


1 gm of 


ill total 








the 


urine ash 


urine 


0—1 . 




1880 




1.23 


11 


1 — 2 . 




1800 
1620 




0.31 
0.31 


2.8 


2—3 . 




2.8 


3—4 . 




1670 




0.26 


2.4 


4—5 . 




1.540 
1860 




0.29 
0.25 


2.7 


5—6 . 




1.8 












in total 












faeces 


0—1 . 




— 




— 





1—2 . 








— 


7.0 


2—3 


5.6 


3—4 . 




1.8 


4—5 . 




— 




— 


l.l 


5—6 . 








in the urine and 15.5 per cent in the faeces. Similar results were obtained 
in other cases. Table 2 shows the results obtained when the radioactive 
])hosphorus was injected into the blood of the same patient. Within 
days 20.5 per cent was lost through the kidneys and 2.5 per cent through 
the gut. Thus about 1/8 of the phosphorus atoms eliminated from the 
blood pass through the gut. By combining the above results it follows 



]5(5 



ADVENTUBES IN RADIOISOTOPE RESEARCH 



that of the phosphoriis found in the faeces about 20 per cent was not 
undigested material but was phosphorus which had already had a share 
in building up the organism and had left it by entering the digestive 
liquids and thus getting into the faeces. 

In the case above, 22.3 per cent of the radioactive P left through the 
kidneys within 6 days and in other cases values varying between 20 
and 25 per cent were obtained. 



Table 2. — Radioactive Phosphori s Injected 
INTO the Blood of a Patient 













Percentage of 


original 


Nub 


ill 


31' of days after 
injection 


Diuresis 
in gin 


radioact 


ive P 




in 1 gm of 


in total 












the ash 


urine 


0—1 








1650 


0.78 


12.5 


1—2 








1510 
1S50 


0.20 
01.5 


3.1 


2—3 


i 






2.9 


3—4 








4—5 








850 
1450 


0.16 
0.13 


1.6 


5— R 


. 






2.2 


7 — S 








SOO 
2000 


0.09 
0.10 


0.6 


8—9 


, 






1.8 












in 1 gm of 


in total 












the ash 


faeces 


0-1 








— 


0.085 


0.24 


1 — 2 








— 


0.11 
0.072 


1.37 


2—3 









0.37 


3—4 








— 


0.072 


0.56 


4—5 








— 









In carrying out experiments like those described above, the most 
satisfactory procedure would be to replace by radioactive labelled 
phosphorus atoms the normal phosphorus present in all the foodstuffs 
administered. By bombarding the material in question with a strong 
source of slow neutrons w^e could turn some of the phosphorus atoms 
into radioactive phosphorus; but such a process always leads to a dis- 
ruption of the molecular bonds of the phosphorus atoms which become 
activated and so to a destruction of the chemical compound. We must 
therefore content ourselves with adding inorganic radioactive phosphate 
to the food consumed and try to obtain a mixture of radioactive inor- 
ganic phosphate and food as uniform as possible. In our experiments 
carried out with human suljjects the sodium radiophosphate was admi- 
nistered in a large volume of milk. Milk contains 0.0795 per cent of 
inorganic phosphorus and about half that amount (0.036 per cent) 



STUDIES OX THE METAHOLISM OF PHOSPHOKVS IX AXIMAT.S 157 

of phosphorus in organic form. Although the ]a11(M' does not exchange 
^vith the atoms of the inorganic radioactive phosphate, the l)ulk of the 
j)hosphorus (0.0795 per cent) reaches a state of kinetic equiUbrium 
with the radioactive phosphate added and l)ecomes radioactively indi- 
cated. During the digestion process the 0.036 per cent will be set free 
from its molecular binding and only at this stage will it have an oppor- 
tunity to become thoroughly mixed (in an atomic sense) with the radio- 
active phosphate atoms. While, as has already been mentioned, it would 
be preferable in investigating phosphorus metaboHsm to utilize food 
in which all the phosphorus atoms are labelled, it is not probable that 
the information obtained with such material would be appreciably 
different from that obtained in the experiments described in this paper. 
Experience shows that the retention of phosphorus docs not depend 
on the form in which the phosphorus is present^ in the food, on whether 
it is present as inorganic and thus exchangeable phosphate or as non- 
exchangeable. Ducks reared on diets containing phosphate only in inor- 
ganic form matured normally and laid 85 to 795 eggs during the first 
summer^. About 15 per cent of the phosphorus present in nu^at. more 
than half that present in milk, and the greater part of that present in 
vegetables, i.e. the bulk of the phosphorus eaten, is present in inorganic 
and thus exchangeable form. 

Rats are inclined to eat their offspring and they could easily be fed 
on young rats born by a mother fed on radioactive phosphorus, but 
the chief source of phosphorus would in this case, too, be inorgani(; 
phosphorus, namely that present in the skeleton. 



ELIMINATION OF PHOSPHORUS BY RATS 

We carried out numerous experiments with rats which were fed on a 
normal diet to which radioactive phosphorus was added. In some cases 
we added 0.1 mgm or less in the form of sodium phosphate dissolved 
in a few drops of water which was then soakerl up by a small piece of 
bread given to the animal. The average of several experiments gave a 
total excretion of 26 per cent through the kidneys and of 32 per cent 
through the gut. In some other experiments calcium ]>hosphate was 
administered, mixed with butter, which was given to the rat on a small 
piece of white bread. The result of such an experiment is seen in Table 3. 
which contains the results of the analysis of the urine and the faeces 
collected during 19 days. The urine was concentrated by evaporation, 
ti'eated with fuming nitric acid, and ignited; a known frac-tion of the ash 

iM.,Spkir8 and H. C. Sherman, J. Nutrit. 11, 2ir) (1036). 
-G. FiNGERLiNG, Biochem. Z. 38, 44<S (101 1). 



15(S ADVENTURES IN RADIOISOTOPE RESEARCH 

obtained was then introduced under the Geiger counter. 19 days later 
the rat, which weighed 256 gm, was killed, the corpse was treated 
with fuming nitric acid to destroy organic compounds, the fatty residue 
was treated with cone, sulphuric acid, and then ignited in an electric 

Table 3. — L.jmgm Radioactive Calcium Phosp- 
hate ADDED TO Normal Diet of Adi'lt Rat 



Number of days after 
taking rad. P 



Percentage of original rad. P 



in the urinu in the faeces 



11.4 13.1 

3.9 4.7 

2.7 2.4 



0— 3 

3— 7 

7 — 10 

10—13 j 1.8 0.93 

13—16 

16—19 



Total . . 



1.3 1.1 

1.2 1.88 



22.3 24.0 



' Faeces contaminated by urine. 



oven. 50.2 per cent of the phosphorus given was found in the ashes, 
which were to a large extent composed of calcium phosphate, and had 
a total weight of 5.84 gm. 

In some cases we added large amounts of calcium phosphate contain- 
ing active phosphorus to the diet. When for example 18 mgm of phos- 
phorus as calcium phosphate were given — this corresponds to about 
four times the phosphorus present in the normal diet — 41 per cent 
of the active phosphorus was eliminated through the gut in the course 
of 19 days and only about 10 per cent through the kidneys. Furthermore 
an analysis of the active phosphorus content of the corpse and the 
excreta revealed that when large amounts of phosphorus were added 
to the diet the animals would eat only part of it, however, carefully 
it was administered. We decided therefore to study the effect of the 
intake of large amounts of phosphorus on dogs. 

The phosphorus atoms absorbed have ample opportunity to enter 
into kinetic exchange with the phosphate ions present in muscles, 
bones, and other organs and also to a certain extent to enter organic 
molecules and replace the phosphorus atoms present there. Many of the 
last mentioned processes are dependent on enzymatic action. The rate 
at which the active phosphorus enters the blood corpuscles, the parti- 
culars of this process, and the distribution of the radioactive phosphorus 
between the blood and the different organs were investigated by Pro- 
fessor LuNDSGAARD and one of us and the results will be published 
shortly. 



STUDIES ON THE METABOLISM OF I'HOSPHOIUS IX ANIMALS 



\y.\ 



PHOSPHORUS EXCHANGE IN ADULT RATS 

A preliminary investigation revealed the following distribution in 
adult rats killed three weeks after eating the radioaelive phosphate 
administered in the lorin of (1.5 mgm sodium phosphate added to the 
normal diet. 

Table 4. — Distribution of Rad. P 

IN Adult Rats Killed 3 Weeks 

AFTER Eating it 



p. c. rad. 1' 



Uiiiic 26.3 

Faeces i 31.S 

Skeleton | 24.8 

Muscles and fat 17.4 

Liver 1-7 

Brain and Medulla 0.1 

Kidneys and Pancreas .... | 0.1 



In interpreting the results obtained it is convenient lo compare the 
radioactivity of equal weights (say 100 mgm) of the ashes, of the bones, 
the teeth, the liver, and so on. These all contain about the same percent- 
age of phosphorus (17 per cent, 17 per cent, 16 per cent); the phos- 
phorus content of the ash of the blood is rather different, but as was 
stated above the behaviour of the active phosphorus in the blood was 
not investigated to any great extent in the course of this work. 

In a series of experiments we gave the same amount of radioactive 
phosphorus to 6 rats. One pair of rats was killed after one week, a second 
pair after two weeks, and a third pair after three weeks. The results 
are seen in the following table. 

The weights of the different skeletons vary to an appreciable extent; 
the weights of the animals were 225, 210, 200, 215, 235 and 220 gm 
before, and 220, 205, 200, 205, 235 and 220 gm resp. after Ihe experi- 



Tablk r, 



AjiinuU killeil weeks after 
eating rad. P 



p. I', of rad. i' found 



in the skeleton in the incisors 



1 


34.2 


2.1 


1 


3r,.3 


2.1 


•> 


32.2 
27.2 
24.tj 


2.8 


•J 


2.1 


3 


2.S 


3 


25.4 


2.7 



160 



ADVEXTURES IX RADIOISOTOPE RESEARCH 



ment. In comparing the rad. P content of different organs of the same 
rat we are independent of the assumption that all the rad. P given 
was actually eaten by the animal, though we are not, when we compare 
the rad. P content of organs from different rats. The greater rad. P 
content of the bones of the animals killed after the lapse of only a week 
cannot, however, be clue chiefly to such a reason as this, because in that 
case the rad. P content of the incisors would also be appreciably higher 
in the case of rats killed after the lapse of one week. This is not the case, 
as can be seen from the figures in Table 5. We must therefore conclude 
that the rad. P taken up by the bones, and in exactly the same way 
all the phosphorus taken up by the bones, has a certain chance of being 
lost again. Indeed an uptake of phosphorus atoms by the bones of an 

Table G 



p. c. of rad. P 

taken, present 

ill 100 mprm 

of ashes 



weight of 

aslies of the 

organ in 

mgm 



p. c. of rad. P 

taken, present 

in the total 

ashes 



a) V a t 


k 


tl 1 


ed 










a f t t" 


I- 


1 


We 


ek 








Bones . . 










O.S 
0.2 
1.3 
3.2 


4300 
100 
253 
1031 


34.3 


Molars 


0.2 


Incisors 










3.3 


Liver . . 












h) V a t 


k 


ill 


ed 






a f t e 


r 


2 


We 


e k s 








Bones . . 


, . 


. . . 


. . < . 





0.7 


4200 


29.5 


Molars . 




. . . 


.... 




0.2 


100 


0.2 


Incisors 










1.9 
2.0 


215 
210 


4.1 


Liver . . 










4.2 







adult rat can only be explained by a corresponding process in the opposite 
direction. Another example of the decrease in the active phosphorus 
content of the bones with time is seen in Table 6. 

While the bones show a decrease in their rad. P content with time 
and the molars no change to within the accuracy of experiments, the 
incisors sliow a marked increase. The incisors of adult rats show a very 
pronounced growth. The discussion of their behaviour is therefore better 
postponed and will be dealt with in the next chapter, where experiments 
on young rats are described. 

The results of an experiment carried out with two rats both killed 
after 5 days time are seen in Table 7. 



1 The weight of the ashes of the liver was found to be very variable. 



STUDIES ON THE METABOLISM OF PHOSPHORUS IN ANIMALS 



1f)l 



T.VBI.K 7 





p. c. rad. P taken found in 

loii nitrin of ushcs 




I 11 




i.;j 1.4 


\IolflT^ 


0.24 0.34 




2.4 2.3 




2.7 1.7 


Muscles 

Brain 


1.7 1.8 
0.46 0.58 



As is seen from the above figures the muscles show a somewhat larger 
content of rad. P than an equal weight of the bones. The active I^ 
content of the brain ash is decidedly lower. To ascertain if the phos 
phorus atoms present are not acid soluble, phophorus compounds are 
also replaced by active P atoms, and the brain treated with 6 per 
c(>nt trichloracetic acid solution. By this means all the acid soluble 
phosphorus was removed. The operation was carried out with great 
care. After igniting the filtrate and residue, the activity of both 
Tractions was measured. We found both fractions to be active, the 
activity of the phosphatide fraction being about i/a of that of Ihc 
trichloracetic acid extract. We are engaged in following up this point 
in greater detail, using more trustworthy methods of separation. 

EXCHANGE OF PHOSPHORUS BY GROWING RATS 

The uptake of phosphorus shown by different organs of rats about 
2 weeks old is seen in Table 8. The rats were killed three days after being 
fed with radioactive phosphorus added to their normal diet. 

Focusing our attention first on the bones we notice that 100 mgm 
of ash contain more than ten times as much radioactive phosphorus as 



Table 8 





Rat I (weight L'7 nm) iliit 11 (vwi^jht '-'4 t,' ii i 




p. c. of rad. !• p_ ^, of ,..^j |. 
weight of ashes ^^^^^ present vveiglit ot ashi'>; ^^^en present 
in mgm '" ^^^ ™^'" '" "^g™ iu 100 mgin 
of ashes „{ ^shcs 


Bones (Leg) 

Incisors 


65.4 10.5 59 10.9 

— 5.8 — 5.8 


Molars 


39 4 2 9 33.8 2.8 


Muscles 


— 11.0 — — 


Blood 


— 2.8 — 2.6 



11 H 



evssy 



162 ADVENTUKES IN RADIOISOTOPE EESEAECH 

was found in the case of adult rats. The high radioactivities of the bones 
are due to the fact that in this case an appreciable part of the bones an^ 
actually grown from blood of high radioactive phosphorus content ; 
a rapid formation of new cells takes place, in whose building up radio- 
active phosphorus participates. 

A very conspicuous difference is found between the active phosphorus 
content of the molars of rapidly growing and of adult rats, the great 
difference being due primarily to the low^ exchange values in the 
latter. 

The brain as a whole was found to contain 0.5 per cent of the active 
phosphorus taken by the animal. 

The ratio between the rad. P content of the muscles and the bones 
is nearly unity in the case of the young rats, while in adult rats the 
muscles show a higher rad. P content. 

When we compare the radioactive phosphorus content of the bones 
of growing rats, we find for example more activity in 100 mgm of the 
ashes of the bones of animals killed after one week than in those killed 
after two weeks. This is due chiefly to the fact that the phosphorus atoms 
present in the bone at a certain time will soon be found in an entireh' 
new part of the growing skeleton, and will also have a certain chance 
of leaving the skeleton entirely. If we want to obtain information on the 
latter point we must compare the "radioactive" phosphorus contents 
of whole skeletons. We carried out such experiments, comparing the 
whole of the leg material. Five very young rats having a total weight 
of only 25 gm were fed on their normal diet plus some radioactive 
phosphorus (0.50 mgm each). Two were kihed 2 days later and three 
65 days later. 10 mgm of the ashes of the leg bones of animals killed 
after 2 days contained 8.4 times as much radioactive phosphorus as 
that of rats killed after 65 days. The active phosphorus atoms were 
in fact distributed all through the greatly increased amount of bone 
tissue ; the leg bones increased in the course of 63 days to about ten times 
their original weight, as can be seen from Table 9. When we compare 
the radioactive phosphorus content of the total bone material of the 
legs, the difference between the rats killed after 2 days and after 65 
days is much less ; the difference still present is due to the loss of phos- 
phorus atoms by the bone material. The phosphorus atoms which were 
present in the bone for a while and left it again will be found partly 
in the excrements but to some extent also in some of the organic 
compounds building up the organism. In the course of two months 
about one third of the phosphorus atoms originally present left the 
skeleton. 

A comparison of the behaviour of the active phosphorus present 
in the incisors with that in the bones is difficult in view of the rapid 
using up and replacement of the incisors. Prof. Holst, Prof. Krogh 



STl'DIES OX THI-; Min'AHOLIS.M OF PnOSPHORlS l.\ AM.MALS 



163 



and one of the Avrilers of this paper are at present engaged on an 
investigation of the exehange of phosphorus in tho incisors on differ- 
(Mil hnes. 



Tahi.k n 





I'eriod 


bl-t 

tivc 


]' ;nul kill 


of 
III,' 


Weight 

of bone ash (legs) 

iu mgm 


p. c. of 

radioactive 1' 

present 


•1 


(lays 

?9 
99 










65.4 
59.0 

440 

514 

<)13 


7.4 


9 










1 .■) 


(i5 










4.1 


(i5 










5.1 


fi5 










5.5 















UPTAKE OF PHOSPHORUS IN PREGNANT RATS AND IN HUMAN 

PLACENTA 

InTaljle 10 the result of the investigation of adult normal and pregnant 
rats is seen. Those designated 1 were killed after a lapse of one week, 
those marked II after two weeks. 

As can be seen from the above figures the different organs of the preg- 
nant rats took up less rad. P than normal rats, the difference being 
found at least partly in the foetus and placenta. In the first rat, which 
was in an advanced stage of pregnancy, the foetus and still more the 
placenta had a high content of rad. P, higher than any organ of the 
mother. We find here again a very conspicuous illustration of the differ- 
ence between the taking up of P through an exchange process and through 

Table 10 





Xcinnal rut ji. c. 
of rad. P taken 

present in 
100 mgm ashes 


Pregnant rat p. c. 
of rad. P taken 

present iu 
100 mgm ashes 


I Bones 

II Bones 

I Incisors 


0.78 

0.74 

1.3 

1.9 

0.21 

0.23 

2.0 

1.94 


0.49 
0.52 
1.2 


II Incisors 


1 7 


I Molars 


12 


II Molars 


It) 


I Liver 


1 () 


II Liver 


1.0 


I Foeta 


2.7 


II Foeta 


0.54 


I Placenta 

II Placenta 


4.0 
2.3 







ir 



164 ADVENTUKES IN KADIOISOTOPE RESEARCH 

actual growth, the latter being much more effective in introducing 
rad. P into the tissue. An appreciable part of the foetus has actually 
been built up by utilising the circulating rad. P and has correspond- 
ingly a high ^^P content. This is still more the case for the rapidly growing 
placenta. In the case of the second animal, pregnancy occurred at a 
much later date than the intake of rad. P. The foetus was nourished 
by blood poor in rad. P, and correspondingly the rad. P content of 
the ash of the foeta was much less. Whereas in the first case the 
weight of all foeta was 345 mgm, in the second case it was only 52 
mgm, the weight of the placenta ash being 43 and 12 mgm res- 
pectively. 

We also had an opportunity to find what was a comparatively very 
high rad. P content for the placenta of a human subject; as much as 
■0.095 per cent was found in the ash of the placenta, which weighed 
133.8 mgm. We can estimate the total ash which the patient in question 
should give on ignition as 2800 gm. The weight of the placenta ash 
thus amounted to less than V20000 ^^ ^^^^ total ash, while the rad. P con- 
tent was as much as i/^^j^ of the total amount of rad. P given, showing 
a concentration of rad. P in the placenta ash more than twenty times as 
great as that in the average ash of the body. One might try to explain 
the high rad. P content of the placenta by its high blood content. That 
this explanation fails is seen, however, from the following. The ash 
of the placenta was found to weigh 133.8 mgm and the ash of about 
5 cc. of blood would weigh the same. But as early as 8 hours after the 
injection of rad. P such a volume of blood was found to contain less 
than Vioooo ^^ ^^^ latter^ and after the lapse of a few days — when the 
placenta were removed — still less. The high rad. P content of the pla- 
centa cannot therefore l)e due to their blood content. No activity could 
be detected in the ash of the few weeks' old foetus removed in the course 
of an operation, but the weight of this sample amounted to only a few 
mgm. 

UPTAKE OF PHOSPHORUS BY RACHITIC RATS 

We carried out a set of experiments on two months' old rachitic rats, 
which had been used by Frederica and Gudjonson in their experi- 
ments on the effect of vitamin A and D deficiency on rickets. The rats 
were fed before and during the experiments on a diet free from or poor 
in vitamins A and D. The weights of the animals before the experiment 
were 89, 83, 85, 93, 90, 95 and 103 gm. The results are seen in TabJp 11 

1 In the case of another subject wo found 1 cc. of blood to contain 0.0027 pcM- 
cent of the phosphorus injected after the lapse of 12 hours, the blood corpusc^les 
containing 11 times as much active phosphorus as the plasma. 



STUDIES ON TTIE METABOLISM OF I'lIOSPHOKl S IN' ANIMALS 



165 



Tahm; 11 



KilkMl 



Jj. c. froiu tlic rM(l. I' lukcii 
foiiiiil ill 1(111 iiigm iislies 



Bones 



Incisors 



Mclar.-5 



Liver 



\\'ei(^lit ill mem 



Bones' 
I (legs) 



Incisors Molars 1 Liver 



I Week 

1 „ . 

2 Weeks 
2 

3 

3 ., 
3 „ 



4.2 


3.8 


0.7 


3.2 


358 


100 


72 


4.2 


3.8 


1.1 


5.9 


329 


105 


7B 


3.0 
3.5 


4.1 
3.7 


0.9 


5.0 
5.0 


403 
3()1 


113 


55 
(54 


2.7 


5.0 


1.4 


l.S 


313 


115 


H9 


2.2 


3.0 


1.1 


1.2 


419 


109 


57 


2.9 


4.3 


0.9 


1.8 


422 


115 


81 



135 
103 

86 

84 

1(58 

145 

205 



The above l)one figures show a marked dii'ference as compared with 
normal rats of the same age (cf. Table 6). We are engaged in carrying 
out further experiments on rats with rickets. 



GENERAL CONSIDERATIONS 

The rapid entrance of the labelled phosphorus into the bone is in no 
way puzzling. If solid calcium phosphate, one of the chief constituents 
of the bone, is in contact with the solution containing labelled phosphate 
ions a rapid distribution of the latter takes place between the surface 
of the solid phase and the liquid phase, as was seen from the following 
experimeni . 3950 gm freshly precipitated Ca3(P04)2 were shaken 
with 5 cc. of water saturated with C'a3(P04)2 at room temperature 
and containing an infinitely small amount of labelled sodium phosphate. 
After lapse of four hours 84.1 per cent of the labelled phosphate ions 
were found in the solid phase and only 15.9 per cent in the solution. 
The calcium phosphate of the bone tissue being in a very intimate con- 
tact with the blood stream, i.e. with cells containing labelled phosphate, 
a similar exchange to that described above will take place between 
the unlabelled phosphate of the bone and the labelled phosphate present 
in the liquid phase. 

Beside the above mechanism we have to consider two others jusi 
as important. During growth, the bone tissue formed will be buill 
up from labelled phosphorus as long as the blood stream contains 
the latter. 

Finally we have to envisage a third possibility, namely the entrance 
of labelled phosphorus into the bone through a constant break-down 



1 The weight of the total skeleton is obtained by dividing the figures obtained 



for \hv legs by 0.2(>. 



166 ADVENTURES IN RADIOISOTOPE RESEARCH 

of the bone tissue already formed and the formation of new tissue in 
the case of adult animals as well. 

The following examples may help to make the three ways of entrance 
of the labelled atoms into the bone easier to understand. 

1) When solid salts are in contact with labelled ions of the solution 
within a short time a distribution equilibrium of the labelled ions between 
the surface layer of the solid and the solution will take place, as is seen 
for example in the experiment described above. This phenomenon was 
studied extensively by Paneth and his collaborators^ in the case ol 
lead salt which were shaken with solutions containing labelled (radio- 
active) lead ions. 

2) If we deposit for example lead electrolytically from a solution 
containing labelled lead ions, the metallic deposit will be a labelled 
one, just as the bone grown from blood containing labelled phosphorus 
will contain labelled phosphorus. 

3) In investigating the exchange between metallic lead and a solution 
of labelled lead ions, or vice versa, we find'^ a different behaviour to 
that described above in the case of lead salts. The exchange in the case 
of metal is not restricted to the uppermost atomic layer of the lead 
surface; many atomic layers are involved in the exchange process. 
This is due to the fact that the lead actually goes into solution from 
certain parts of the surface, while lead ions are discharged, at other 
parts. This is a much more effective process in bringing about an exchange 
between the lead atoms in the solid and in the liquid phase than that 
observed in the case of solid salts where only the uppermost atomic 
layer is involved (within any resonable time) in the exchange process. 
The entrance of labelled phosphorus into the bone will also be much 
facilitated if it is not only the uppermost phosphate layer that is invol- 
ved in the exchange process; if in fact the bone is destroyed at certain 
places and rebuilt at others. In view of the important enzymatic actions^ 
going on in the bone tissue such a reversible breakdown process will 
easily occur. 

Summary 

By adding radioactive phosphorus (phosphate) to the diet of rats, the 
metabohsm of the phosphorus atoms taken in with the diet can be followed 
np in the animal body. An appreciable part of the phosphorus taken finds 
its way not only in growing but also in adult animals into the t)ones, teetli, 
muscles, and different bodily organs. 



IF. Paneth and W. Vorwerk, Z. phij,s. Chem. 101, 445, 480 (1922). 
2G. Hevesy, Phys. Z. 16, 52 (1915) 

3 R. RoBisox, The Significance of PhospJwric Esters in Metabolism. Xew York 
(1932). 



STUDIES OX THE METABOLISM OF PHOSPHORUS IX AXIMALS 167 

In growing animals it was found that the atoms alroadv present at an 
early stage of the formation of the skeleton beeome distiibuted in the 
eourse of time over the different parts of the skeleton and other organs 
demonstrating thus the dynamical nature of the building up of bono tissue. 
Some of the phosphorus atoms present in the bones leave the skeleton for 
good, being eliminated through the kidneys or the bowls or becoming located 
in other organs of the body. 

The replacement of individual phosphorus ato rs by other ])liosphorus atoms 
also takes place in the bone tissue of adult animals including that of the 
teeth. 

It was ascertained that about one-seventh of the phosphorus found in 
the faeces of a human subject is due to material which has entered the 
intestines through the digestive juices after being located in the blood stream 
or in the organs of the body for a shorter or longer tim(\ 



Origiinlly published in Kgl. Danske V idenskahernes Selskab. Biologiske Meddelelsei- 

13, 13 (1937) 

20. INVESTIGATIONS ON THE EXCHANGE OF 

PHOSPHORUS IN TEETH USING RADIOACTIVE 

PHOSPHORUS AS INDICATOR 

G. Hevesy, J. J. IIoLST and A. Krouh 

From the Institute of Theoretical Physics, Dc ntistiy School and Zoophysiological 

Laboratory, Copenhagen 

ANATOMICAL INTRODUCTION 

The hard part of a tooth is composed of three distinct substances viz. 
1he dental substance proper, dentine, the enamel, and the cement. 
The dentine constitutes by far the largest portion; the enamel is found 
in a comparatively thin layer partly covering the dentine; and the ce- 
ment covers the surface of the root in a thin layer. In the case of the 
canines of cats we found the weight of the enamel ash to be 11.2% of that 
of the dentine ash, the weight of the enamel before ashing being equi- 
valent to about 9.7% of that of the dentine. 

The dentine is penetrated throughout by fine tubes (dentinal tubes) 
starting from that side of the dentine which faces the pulpa cavity; 
they have an initial diameter of 2 to 8// and do not much diminish 
in size at first as they approach the surface; the distance between adja- 
cent tubules is about two or three times their width. From the tubules 
numerous immeasurably fine branches are given off and penetrate 
1 he hard intertubular substance. Near the periphery of the dentine, 
the tubules, which by division and subdivision have become very fine, 
terminate imperceptibly in free ends. It is reported that tubules have 
been observed passing into the enamel in the teeth of marsupial animals, 
and to a less marked degree in human teeth. In this case they pass, not 
into the enamel prisms, but into the inter-prismatic substance. The ena- 
mel is made up of microscopic columns, very hard and dense, arranged 
close side by side, and fixed at one extremity on to the subjacent sur- 
face of the dentine. The enamel columns have the form of six-sided 
prisms. Their diameter is about 0.005 mm. They are united by a small 
amount of substance which appears to be similar to the intercellular 
substance of an epithelium. The small amount (about 1%)^ of 



' 1. H. Bowos and :\I. M. Muray, (1935) Biochem. J. 29, 12, 2721. 



KXOHA.NGE OF PnOSTHOKTS TX IKETll 



] (V.) 



organic mat tor in 1h(^ c^namel is probably found 1o a large extent 
in the above mentioned connective substance. Jn marsupials and 
some rodents there are regular eanaliculi in the interprismatic sub- 
stance. 

The central cavity of a tooth is occupied by a soft and very vascular 
dental pulp, containing cells, blood-vessels, nerves, and fine connective- 
tissue fibres. The cells are partly disseminated in the matrix and partly 
form a stratum at the surface of the pulp. These superficial cells, the 
odontoblasts, send out elongations into the tubules in the dentine. It is 
through the intermediary of the pulp that constituents of the blood get 
into the hard tissues of the teeth. 



Chemical composition of the teeth 



a) Dentine. 



On analysing a great number of dry human dentine samples Bowes 
and Murray^ found a loss in weight of the fresh tissue on ignition 
amounting to 29 — 29.7%. The losses on ignition found in some of 
our experiments can be seen in Table 1, in which we have also 
included for the sake of comparison the values found for the tibia 
and jaw. 



Tablk 1. Albino Hat 2(Hl g 



Organ 


Loss on ignition 

in % of fresh 

weiglit 


( Proximal oikI 

Incisors Oistal end 


33.0 
25.0 


.VvtM-at^e 

Molais 


2b.4 
27 


( llt-ad 


79.1 


Tibia , 

1 .Aveiage 

(Jat 4 kgm 

Incisors 

Canine 


(33.2 

32.0 
3.") 


Molar 


3S.0 


,Jaw 


.")(». 4 


Tibia epiphysis 

Tibia diaphysis 


()6.8 
36.7 



The average values found for the chief constituents of the dentine by 
Bowes and Murr.w^ are seen in Tabic 2. 



1 I. H. Bowos and y\. ^\. Mt^ray, (193(i) Biochem, J. 30, 1?)77, 



170 



ADVENTURES IX RADIOISOTOPE RESEARCH 



Table 2. Analyses of Dentine of Human Teeth 
(% in Dry Dentine) 



Slight hypoplasiii Severe hypoplasia 



Ash 
Ca . 
P .. 
CO2 
Mg 
CI . 



71.09 


70.28 


27.79 


26.96 


13.81 


13.5 


3.18 


3.10 


0.835 


0.728 


— 


0.023 



Bowes and Murray give the following average figures for the compo- 
sition of the enamel: 



Table 3. Analyses of Enamel of Human Teet 



H 



Slight liyiioplasia Severe h^-poplasia 



Ash 
Ca . 
P .. 



95.38 
37.07 
17.22 



94.67 
35.81 
17.72 



Slight hypoplasia Severe hypoplasia 



CO, 
Mg" 
CI . 
Fe 



1.952 
0.464 
0.3 
0.25 



2.434 
0.477 
0.19 



As is seen from the above figures phosphorus is the second most 
abundant mineral constituent of the teeth, its share in the dentine 
amounting to 13.5 — 13.8% and in the enamel to 17.2 — 17.7% while 
in the dentine ash 18.2—18.4% in that of the enamel 18.4 — 19.4% 
w^ere found. In the ash of the incisors of rats an even higher phosphorus 
content of 20% was found. Bone ash contains an only slightly lower 
amount of phosphorus than tooth ash, the values found varying betw^een 
17.9 and 18.5%. 

In distinction to the chief constituents of the teeth the minor consti- 
tuents vary within wide limits. The composition of the mineral consti- 
tuents of the teeth corresponds approximately to a mixed crystal of the 
minerals hydroxide-apatite and carbonate apatite, the former predomi- 
nating strongly. As in apatite minerals the OH ions of the tooth apatite 
can be replaced to a certain extent by F ions for example. The degree 
of replacement of 0H~ by F^, will depend primarily on the fluorine 
content of the blood during the development period of the tooth and 
also on that which circulates in the fully calcified tooth. The fluorine 



EXCHANGE OF PHOSPHORUS I\ TEETH 



171 



o 



content of the blood will depend on the fluorine content of the food and 
water taken up. It is thus easy to explain why the fluorine content of the 
tcH'th varies within wide limits (see Table 4). The high fluorine content 
f 1he teeth of human beings living at Colorado Springs is due to the 
high fluorine content of the water which amounts to up to 2 mgm per 
Uler. The high fluorine content of the teeth of some North African 
sheep is to be explained by the high fluorine content, above 0.02%, 
of the soil on which they graze. On such soil plants of high fluorine 
content grow, are eaten by the sheep, and lead to an abnormally high 
fhiorine of the blood plasma, which in turn leads to an abnormally high 
I'eplacement of OH^ by F in the teeth^' 2. 3- 4 



Table; 4. Flt'orink Context of Teeth Ash 



Mani 

Marine animals 
Rats2 



Man^ New York 



Man New York 

Man^ Colorado Springs 

Man Colorado Springs 

Man* 

Man* 

Calves* 

Calves* 

Sharks* 

Sheep, young from neighbourhood of Norwe- 
gian ahiminium factory where fluorides are 
utilised* 

Sheep^ North Afric-a 

Sheep^ North Africa, attacked l)\- fluorine 
disease 





/o 


teeth 


0.03 


teeth 


0.69—0.74 


teeth 


0.006—0.03 


dentine 


, 0.065 


enamel 





dentine 


0.1 12 


enamel 


0.065 


dentine 


0.030 


enamel 


0.005 


dentine 


0.022 


enamel 


0.0057 


teeth 


0.89 


incisors 


0.45-0.49 


teeth 


0.04 


teeth 


0.32— 0.4 ■> 



The much higher fluorine content of animals living in sea watci is 
also due to the comparatively high fluorine content of the latter. In 
1 he same way that F replaces OH in the apatite lattice, magnesium 
for example replaces calcium. Human dentine ash has a magnesium 
content*^ of 1.18 — 1.39% whereas human enamel ash has only 0.42%. 
While the calcification of the tooth tissue is presumably the result of 
specific cell activity, it is quite possible that later on a replacement of 

(DR. Klement, Naturwiss. 21, 662 (1933). 

(2)R. R. Sharpless and E. V. McCollum, J. Xutnt. 6, 163 (1933). 

'3)H. Boi-ssEVAiN and W. F. Drea, J. Dent. Bes. 13, 495 (1933). 

(*)K. RoHOLM, Fluorine Intoxication, p. 260. Copenhagen (1937). 

(5) M. Gand, a. Chavnot and M. Langi.ais, (1934.) Bull. Innt. Ili/g. Maroc 

Xos. I-II. 
(«)M. M. Murray, Biochem. .7. 30, 1568. (1936.) 



172 ADVENTURES IN RADIOISOTOPE RESEARCH 

calcium by, for example, magnesium takes place, governed chiefly by 
solubility (chemical affinity) conditions, and it is quite conceivable 
that in the course of time more and more calcium is replaced by magne- 
sium if the magnesium: calcium ratio is in favour of such an exchange. 
The enamel being characterised by a decidedly poorer lymph circulation 
than the dentine, the much lower magnesium content of the former can 
easily be accounted for by a reference to the above considerations. 
The 0.42% magnesium found in the human enamel possibly got into 
the latter wholly or to a large extent during the formation of the enamel 
tissue. The presence of as much as -i^j^ of magnesium in elephant den- 
tine is possibly due to a high magnesium content of its food or to a 
high magnesium retention in its blood, ft is also of interest to remark 
that the magnesium content of the teeth found in prehistoric skelet- 
ons is only one third of that found in leeth of recent generations, fur- 
thermore that carious teeth^ show a greatly increased magnesium con- 
tent. Besides the elements discussed above, spectroscopic investiga- 
tion^ revealed the presence of traces of Na, Ag, Sr, Ba, Cr, Sn, Zn, Mn, 
Ti, Ni, V, Al, Si, B and Cu in dental tissue. 

That the concentration of the minor constituents of the teeth does 
not fluctuate between still wider limits is due to the narrow limits 
within which the concentration of most elements in the blood plasma 
is restricted. This is caused partly by a prevention of the resorption 
of excessive quantities of the elements, conspicuously show^n in the case 
of calcium, and partly by prompt removal chiefly through the kid- 
neys of excessive amounts of the mineral constituents present in the 
plasma. But even in spite of this levelling mechanism of the blood 
plasma some of the mineral constituents are deposited to a remark- 
able extent in the tooth tissue, as is seen above in the case of flu- 
orine, and it is quite possible that exon an excessive replacement of, 
for example, the calcium by magnesium, sodium, or potassium might 
lower the resistance of teeth to disease. 

While the conclusions given above are based partly on hypothetical 
assumptions, in the case of lead, which also replaces calcium in the 
crystal lattice, the accumulation in the teeth with time can clearly be 
shown. While small children have only negligible amounts of lead in 
their teeth, the lead content increases with age^, the increase being 
markedly greater in the case of carnivorous than herbivorous animals, 
presumably on account of a greater lead intake in their normal nourish- 
ment. In the case of lead poisoning the lead content of teeth is greatly 



(i) T. Francia, Ann. Clin. Odoniat. 8, (iil.'j, (1931); M. M. Murray uirI 

J. H. Bowes, Brit. Dent. J. 61, 473, (1936). 
^2) E. LowATER and M. M. Murray, Biochem. J. 31, S37, (1937). 
^3) F. Pfrieme, (1934) Arch, f. Hyg. Ill, 232. 



PIXC'UANliE Of I'HOSl'HOIUS I.\ TEETIC 173 

increased. All those observations support the hypothesis, that even in 
fully formed teeth an exehan^c^ of mineral constituents is regularly 
taking place. To test this hypothesis we have studied the exchange of 
phosphorus by means of labelled phosphorus atoms. 



PHOSPHORUS EXCHANGE IN TEETH 

We investigated the movement of the phosphorus atoms both in the 
teeth of fully grown and growing animals by using labelled phosphorus 
atoms as an indicator. By adding radioactive phosphorus, ])reparcd 
from sulphur by the action of neutrons, to food administered to animals 
at a known date, it is possible to distinguish the phosphorus atoms 
which were present in the food sample and which have been retained 
and deposited in the organism, from those already present in the body 
and the teeth at the start of the experiment. We can thus follow the 
movement of the phosphorus atoms taken in for example a glass of 
milk and investigate if and to what extent these particular atoms get 
into the teeth and how they are distributed there. 

The dentine contains 14% and the enamel 17.5% of phosphorus in 
the form of phosphate (PO^). it is the movement of these phosphate 
radicles which we actually investigate. For the sake of brevity we shall 
often use the word phosphorus in discussing the behaviour of the phos- 
phate radicle. We may recall that the phosphorus taken with food, 
amounting in the case of an adult to somewhat more than 1 gm. per 
day, is to a large extent (in most cases up to about 80%) absorbed from 
the gut and gets into the blood stream. Adult human blood contains 
44—50 mgm% of phosphorus of which only 2 — 5 mgm% are present 
as inorganic P. Very different views have been put forward on the for- 
mation of the l)one and tooth tissue, but they all consider the blood 
plasma as saturated or nearly saturated with calcium phosphate and the 
precipitation of the latter from the plasma as being of paramount impor- 
tance for the ossification process. The solubility of calcium phosphate 
in the plasma is very strongly affected by the presence of proteins, carbo- 
nate and bicarbonate ions, and possibly also other constituents. It is 
also dependent on the acidity of the blood, slight changes in which 
may be sufficient to produce precipitation. It seems very probable that 
it is not simple calcium phosphate but a complex salt of the apatite 
type, a solid solution of hydroxide apatite and carbonate apatite, that 
precipitates. 

In addition to the inorganic phosphate, blood contains a phosphoric 
ester at a comparatively high concentration which is mainly found in 
the corpuscles; as it cannot yield phosphate ions by dissociation, this 
ester does not affect the saturation of the blood with respect to calcium 



174 



ADVENTURES IX RADIOISOTOPE RESEARCH 



phosphate. However, as RobisonI discovered, the cartilage and 
osteid contain an enzyme, phosphatase, which hydrolyses this ester, 
thus setting free inorganic phosphate, whereby the concentration of the 
phosphate ions increases and a supersaturation occurs, followed by a 
precipitation of the calcium phosphate in the matric of the tissue. With 
the discovery of the bone phosphatase a second agency (in addition to 
the acidity change) of great importance was found, regulating the cal- 
cium phosphate precipitation leading to ossification. Robison found 
that the enzyme had the greatest activity in ossifying cartilage, bones, 
and teeth of very young animals, the activity per unit weight of tissue 
decreasing with age. Although the plasma contains on an average only 
0.5 mgm of phosphorus present as phosphoric ester per 100 cc. this is 
completely hydrolysable by the bone phosphatase and thus supplies 
phosphate ion amounting to about ^/g of the inorganic phosphorus 
present in the plasma, an amount amply sufficient to bring about a 
supersaturation and a subsequent precipitation of calcium phosphate, 
vjr more correctly of the apatite-like bone substance, from the already 
nearly or fully saturated plasma. The conclusions arrived at in this 
paper are independent of the special mechanism assumed for the ossifi- 
cation process. 



DISTRIBUTION OF LABELLED PHOSPHORUS IN THE INCISORS 

OF RATS 



The rapidly growing incisors of rats are very suitable for studying 
the distribution of phosphorus. According to Friderica and Gudjox- 
SONS^ the average extrusive incisor growth per week is 2.7 mm. in the 
case of adults and 3.4 mm. for young 
rats. As seen in Fig. 1 A the cross sec- 
tion of the pulpa is very large at the 
proximal end and gets narrower toward 
the distal end, the last millimetres of 
the teeth being free of pulpa. The 
problem we have to investigate is how 
the distribution of newly formed cal- 
cium phosphate in the incisor takes place. 
Two extreme cases must be envisaged: 

a) the labelled phosphate is deposited Fig. 1. 




1 R. EoBisoN (1912) The Significance of Phosphorus Esters in Metabolism, 
New York. 

2 L. S. Friderica and S. V. Gudjonsons, Kgl. Danske Vid. Selsk. BinJ. 
Med. 28, 813 (1931). 



exchax(;e of iMiosrnoRrs ix tkkii 



17:> 



in close proximity to t]i(> ])vjlp from which 11 is derived, while the 
tissue formed a1 an earlier date is pushed aloni,^ in lh(> direction of" 
jrrowth; 

b) the labelled phosphate is equally distributed throughout the incisor. 

(Jutting incisors tranversally into pieces and analysing these separa- 
tely revealed the fact that the largest part of the labelled phosphate 
is found in those regions of the incisor where the pulpa is strongly deve- 
loped, but that some of the labelled phosphate is found all through 
the incisoral tissue (Tables 5 and 6). 



Table 5. Distribition of Labelled Phosphorus, Contained 

IN THE Normal Diet. Found in the Incisor after 2 days. Weight 

OF THE Rat 210 gm -f- Denotes Upper — Lower Teeth 



part, of the incisor 


Weight of asli 
ill mpm 


% of labelled V 
taken, found 


';;o of the labelled 
P per mgm ash 


Proximal I -J- 


38.2 
40.8 
20.2 
27.2 
92.() 
115.2 
36.4 


0.42 

0.47 

0.37 

0.38 

0.125 

0.072 

0.008 


0.011 


Proximal Il-f- 

Proximal I -^ 


0.012 
0.013 


Proximal II -^ 


0.014 


Middle 


0.00135 


Distal 14- 


0.00063 


Distal II— 


0.00022 







Percentage of labelled P found in the total incisors = 1.85. Average 
per 1 mgm ash = 0.005. Biggest ratio between proximal and distal 
end = 60. 

Table 6. Distribution of Labelled Phosphorus, Administered 
IN the Normal Diet, found in the Incisor after 7 Days. Weight 

of the Rat 240 gm 



Part of the incisor 



Weight of ash 
in mgm 



Proximal I + [ 25.0 

Proximal Il-f 23.6 

Proximal I -^ 21.8 

Proximal II 4- 31.6 

.Middle+ ' 81.6 

Middled : 68.0 

Distal -f and -f- i 20.3 



% of labelled P 
taken found 



% of the labelled 
,r per mgm ash 



0.28 

0.31 

0.29 

0.32 

0.204 

0.206 

0.020 



0.011 

0.013 

0.(»13 

0.010 

0.0032 

0.0031 

0.00074 



Percentage of labelled P found in the total incisors = 1.69. Average 
percentage per 1 mgm ash = 0.006. Biggest ratio between proximal 
and distal part = 18. 



176 



ADVENT IRES IX KADIOISOTOI'E KESEAKCH 



In the expeiimeiits now to be described the distal part of tlie incisor 
was removed l)y operation one day before labelling the phosphorus 
present in the blood. In these experiments the radioactive P was not 
added to the food but given in the form of subcutaneous injections. 
2 days, 5 days and 8 days after the administration of the labelled phos- 
phorus the end part of the freshly grown incisor was again removed 
by operation and its radioactivity ascertained. The distal parts removed 
were all outside the range of the pulp. The figures obtained are seen 
in Table 7 and those from a similar experiment in Table 8. 







Tablk 


/. 








Days after intake of 


aheUcil 1' 


W'uitrht of the 
tissue iu mgm 


% of tlie labelled 

P found in 1 

mgm fresh tissue 









42.8 
16.4 
20.4 
26.6 


0.00089 








0.00030 


s 






0.00066 


13 


(rat killed ) .... 




0.00090 











Percentage of the labelled P found in mgm of average incisor tissue 
= 0.0076. The removed distal ends contained 8 to 25 times less labelled 
P than the average tissue. 



Table 8. 



Days ufter intake of labelled V 


Wei^lit of the 
tissue in mg^n 


■^^V, of the labelle'l 
V found iu 1 
mgm fresh tissue 


,> 


14.4 
11.0 
21.3 


0.00040 


S . . 


0.00044 


13 (rat killed) 


0.00062 







Percentage of the labelled P found in 1 mgm of average incisor 
tissue = 0.0062. The removed distal ends contained 10 to 16 times 
less labelled P than the average tissue. 

Though the figures in the tables above clearly show that the deposition 
of labelled phosphorus is not restricted to the regions in the vicinity 
of the pulp, but that the labelled phosphorus is to be found even in the 
most remote part of the incisors, we attempted to obtain incisors with 
an appreciably larger pulp-free part. As is well known, rats, being 
rodents, grinfl their teeth and thus continually remove parts of th(> 
pulp-free end of the growing incisors. By eliminating the upper incisors 
the animal was prevented from gnawing and incisors were thus obtained 
in which the distal pulp-free end had a length of 10.5 mm. as shown 



EXCHAXOE OF PHOSPHORIS TX TEETH 



17 



Table 9. Distribition in the Inci.sor after ."} DA'is. Lahelled 

PhOSPHORTS In.JECTEI) .Sl'Bt'rT.\NE()t'.SLY. 

Weight of the Rat abott 2()(t gm 



Part of the incisor 
(coinp. Kitr. 2> 


WeiRtit of 

the tissue 

in nis»i>i 


VVeifflit of 
the iv^li 
ill lutnii 


"r, hilielle.l I' 
f(i\inil per mgin 
tissue 


•^0 hibelled V 

found per niRm 

a.sli 


I (Proximal end) 


13.1 


9.2 


0.0103 


0.0151 


II 


14.5 


11.0 


0.0079 


0.01 Itl 


Ill 


24.0 


17.1 


0.0026 


0.0040 


IV 


1.5.3 


11.0 


((.00021 


0.00030 


V (Distal end) 


12.0 


9.0 


0.000033 


0.000044 



in Fig. 1 B. The result of this experiment is seen in Table 9 and the dia- 
gram Fig 2. 

The content ol' labelled P varies between 0.01% ai the proximal end 
and 0.000033% at the pulp-free distal end, thus diminishing by a factor 



10 mm 




Fig. 2. Distribution of labelled phosphorus in the int-isor of a rat 

killed 3 days after the administration of the phosphoius. 

The figures below give the relative amounts of labelled phosphorus 

present in 1 mgm of fresh tissue in the section in question. The 

figures above give the length of the .section in mm 

of 1/300. On comparing the activity of the ash obtained by igniting 
the incisor the figures work out to be 0.015% and 0.000044% respec- 
tively, corresponding to a factor of 1/340. The average content of labelled 
P in 1 mgm tissue was found to be 0.0041%, in 1 mgm ash 0.0059%. 
Fig. 2 shows both the location of the pulp and the distribution of the 
lal)elled phosphorus. It is seen clearly that the bulk of the labelled phos- 
phorus atoms are to be found in the vicinity of the pulp but an amount which 
is far from being negligible reaches even the remotest part of the incisor. 
In Fig. 2 we have inserted the relative abundance figures of the labellefl 
phosphorus present in the different parts of the incisor. A part of the 
first sector amounting to 1.4 mm. grew during the time which elapsed 
between the injection of the labelled P and the killing of the animal; 
the other parts were present before. Out of 204 parts of labelled phos- 
phorus only 100 were found in the first sector and consequently not 
more than 30 in the part actually grown, the remaining 174 or more 
being at least partly located in the parts present before injecting the 
phosphorus. 



1 2 Hevesv 



178 



ADVEXTUEES IN RADIOISOTOPE RESEARCH 



In seeking an explanation of the presence of labelled phosphorus at a 
very considerable distance from the pulp avc must remember that even 
the most remote incisal part of the tooth contains organic constituents. 
The constituents of the blood plasma penetrate through the latter and 
exchange of phosphate radicles and possibly also some ossification occurs 
in situ, though only to a modest extent on account of the poor circulation 
in comparison with that in the vicinity of the pulp. For part V (Fig. 2) 
we found a loss of weight on ignition amounting to 25% of the 
weight of the tissue dried in a vacuum dessicator; part I lost 29.8%; 
and the average loss in ignition was found to be 27.4%. That bones 
rich in organic constituents, i. e. such in which a comparatively 
effective circulation takes place, take up more radioactive phosphorus 
than the diaphysic bones poor in organic constituents had already 
been found previously in our investigations and is also to be seen 
in an example discussed on page 178. 



PHOSPHORUS EXCHANGE IN GROWING RATS. 

It is tempting to explain the parallelism between the abundance of 
organic substance present in the tissue investigated and the percentage 
of labelled phosphorus present by assuming that the latter is chiefly 
present in the organic substance and not in the calcium phosphate 
of the bone or teeth. This possibility must however be discarded because 
blood weighing as much as an incisor contains after the lapse of few 
days less than 0.01% of the labelled phosphorus taken, while that found 
in the incisors exceeds 1%. In view of the importance of this point we 
tested the effect of the removal of the pulp on the exchange data. The 
experiment was carried out on a young rat which increased in weight 
from 87 to 110 gm in the course of the 5 days which elapsed between 
the subcutaneous administration of the labelled phosphorus and the kiUing 
of the animal. Before the analysis the pulp was removed from the two 
upper incisors and the activity of these incisors compared with that 
of the two lower incisors containing the pulp: we also measured the 
activity of the extracted pulp. The results are seen in Table 10. 

Table 10. 



Lower Incisoi-s -|- pulp 
Upper Incisors — pulp 
Tibia 



Fresh we i slit 



Ash weight 



58.2 

33.2 

800.1 



% of the labelled 
V found per 
mgm ash 



37.7 

22.9 

123.2 



0.0192 
0.0185 
0.0234 



Loss of weight 
on ignition 



35.2<% 
31.0% 

84.6% 



EX('H.\NGK OK THOSPHOJM S IX TKETJI 



179 



The adivily of tlic cxlractiMl pulp was very weak and only amounted 
to about 3"o ol lliat of Ihe ui)per incisors. Should the exchan^re of phos- 
phorus in the ealcium phosphate of the teeth be very small, it is however 
quite possible that the amount of labellcni phosphorus present in the 
])ulp would no longer be negligible (eomp. p. 20). 

While in the experiments described above emphasis was laid on the 
investigation of the remote incisal end, in Ihe following experiment 
we cut the proximal end of the incisor into small pieces and compared 
their activity with that of the distal end. The results are seen in Table 1 1 , 
I denoting ihe united parts nearest to the jaw of all four incisors (comp. 
Fig. 3). 

^1 U 1,1 rn rri 




Fia. 3. Distribution oi" labelled phosphorus in the incisor of a rat 

killed 7 days after administration of the phosphorus. 
The figures below give the relative amounts of labelled phosphorus 
present in 1 mgm of fresh tissue in the section in question. The figures 

above give the length of the section in mm. 2.0 should read 2.8. 

Table 11. Distribution of Labelled 

Phosphorus, In.jected Subcutaneously, 

Ix THE Incisor after 7 days. Weight of the Kat 

ABOUT 200 GM 



i'ui'l ul the 


Leugtli 
in mm. 


Weight oi the 


% of tlie labelleil 


incisor 
(comp. Fig. Z) 


fresh tissue in 
mgm 


P found per 
mgm tissue 


I 


2.8 


7.3 


0.0156 


II 


i.y 


1(5.8 


0.0127 


Ill 


1.2 


21.3 


0.0054 


IV 


1.7 


25.1 


0.0025 


V 


1.1 


28.2 


0.0025 


VI 


10.0 


163.7 


0.00096 



Averag(> % of labelled P per mgm tissue = 0.0028, per mgm ash 
0.0038. 

The investigation of the labelled P content of the head (A), the central 
(B) and lower part (C) of the tibia gave the figures seen in Table 12. 

While in the proximal end of the incisor the phosphorus exchange 
is much greater than in any part of the tibia, the exchange of the average 
phosphorus atoms in the tibia is about 29% greater than that of the 
average P atoms of the incisor; this is due to the fact that contrary 
to the tibia a large part of the incisor exchanges phosphorus atoms 



12* 



180 



AUVEXTURES IX llADIUISOTOl'K llESEARCH 



Table 12. 



Weight of the 

fresh tissue in 

mgm 



^\'eigl»t of 
ash in mgm 



% i>f the labelled 

I* found per mgm 

ash 



A I 131.1 

B 312.4 

C 243.8 




0.0080 
0.002() 
0.0027 



in the course of 7 days only to a small extent. The figures in Table 
12 cannot be compared directly with those obtained from fully grown 
animals for the following reason: the growing animal being much smaller 
the percentage of labelled P obtained for the same weight of the organ 
becomes larger; furthermore growth much facilitates the uptake of 
phosphorus. We can, however, compare the ratio of the labelled P 
content of the incisor and of other organs; the value of this ratio for 
the tibia, for example, is found to be not appreciably different in the 
cases discussed above. As to the labelled phosphorus content of the 
blood, this amounted after the animal was killed to only 0.04% per 
gram of blood; assuming a blood content of 10 cc. the circulation 
contained but 0.4% of the labelled phosphorus administered of 5 days. 



THE EXCHANGE OF LABELLED PHOSPHORUS IN MOLARS 



In contrast to the incisors, molars of adult rats do not grow, so the 
labelled phosphorus found in the latter is due solely to exchange pro- 
cesses; the blood stream circulating through the molar carries labelled 
phosphate ions which enter into exchange processes with the calcium 
phosphate of the molar tissue. Such exchange processes also take place 
in the incisors simultaneously with the formation of new ossification 
})roducts. In the molars of adult animals, however, we encounter chiefly 
the former process; but though growth can be excluded we cannot 
discard the possibility of dissolution of tooth tissue at one place and 
a corresponding precipitation of calcium phosphate at another along 
the boundary between the circulating fluid and the tooth tissue. Small 
fluctuations in the acidity or parathormone concentration of the blood 
are sufficient to cause such a process. The molars of the rat decribed 
on p. 181 showed a content of labelled phosphorus amounting to 0.0013% 
per mgm of tissue and 0.0018% per mgm of ash, which is less than in 
the average incisor. The loss on ignition was found to be 26.9%. We 
thought furthermore that it would be of interest to compare the labelled 
P content of the incisors, molars and skeleton, choosing the tibia as 
representative of the latter. The figures obtained are seen in Table 13. 



KXCHANCK OK VHOSIMIOI? IS IN rKKIH 



IHl 



Tablk 13. 





l.al.i-llcil Clomiil 


Labelleil 1' found 






in 1 int^ui fresli 


in 1 nigm jisli 


Loss in 


Or-ui 


tissue as ii iier- 


as a percpiitafje 


weiglit on 




i;e»tage o£ the 


o£ the amount 


ignition 




amount given 


given 




Incisor 


0.0033 
0.0013 


(». 0(144 
O.OOlS 


26.4% 


.Molar 


/o 
63.20/0 


'^nijiu 


0.0024 


0.0064 





The fresh tissue of the incisors contains more labelled I^ than equal 
weights of either the molars or the tibia, but comparing the ashes the 
tibia has a larger labelled P content than both the incisors and the 
molars. A comparison of the labelled P content of the 
ash is in general preferable to that of the fresh tissue, 
the former comparison giving information about the 
percentage of phosphorus atoms replaced by labelled 
ones. The total P content of the ash of the incisors 
varies between 19.6 and 20.0%, and that of the molars 
and the tibia is only slightly smaller, about 18%. A 
closer analysis of the tibia revealed the parallelism 
already mentioned between the content of organic 
tissue and labelled phosphorus; that is shown in Table 
14. The rat was killed 3 days after the administration 
of the labelled P. 

As seen from the figures of Tables 13 and 14 the 
head of the tibia has exchanged a part of its phos- 
phorus content eight times as large as that exchanged by the 
molars. 

Furthermore we compared the labelled P content of the incisors, 
the molars, and the tibia in the case of a rat weighing 220 gm killed 
1 hour after the labelled phosphorus had been administered by sub- 
cutaneous injection. The results are seen in Table 15. The incisor was 



02 



02 




Fig. 4. 



Table 14. 



Part of the tibia, 
see Fig. 4 


Weight of tlie 

fresh tissue 

in mgm 


"„ c,f tlirlalj,.|leil 

P fouiiil in 
1 mgm tissue 


% of tlii'labclleii 

P found in 
1 mgm asti 


Loss on 
ignition 


a 


21.5.4 
64.4 
78.8 
33.1 
81. .5 


0.0028 
0.0033 
0.0019 
0.001.5 
0.0014 


0.0134 
0.0064 
o'.0033 
0.0028 
0.0034 


70.1 


**i 

b, 


48.3 


■^1 
c 


42.0 


b.> 


47. .5 


2 

a„ 


58.7 


"2 





182 



ADVENTURES IX RADIOISOTOPE RESEARCH 



Tablp: 15. 





Labelled i' foiiiid 


Labelled P found 






in 1 mgm fresh 


in 1 mgm ash 


Loss in 


Org;in 


tissue as a per- 


as a percentage 


weight on 




centage of the 


of the amount 


ignition 




amount given 


given 




Incisor 


0.00046 


0.00062 


26.0 


Molar 


0.00025 


0.00034 


27.4 


Jaw 


0.00125 


0.0020 


36.3 


Tibia head 


0.0024 


0.0077 


68.7 


Tibia residue 


0.00068 


0.0014 


52.7 



out in 5 pieces the labelled P content of which is seen below, I denoting 
the proximal end. 



Table 16. 



Weight of 

the fresh 

tissue in 

mgm 



I 

II 

III 

IV 

V 



Labelled V found 
in 1 mgm fresh 
tissue as a per- 
centage of the 
amount given 




0.0062 
0.0027 
0.00040 







One cc. of blood contained 0.5% of the activity injected; assuming 
a blood content of 10 cc, only 5% of the labelled phosphorus injected 
was present in the circulation after the lapse of 1 hour. Only 1 hour after 
administering the labelled phosphorus the tibia phosphorus was found 
to be 1000 times less active than the blood phosphorus, while for the 
molars the corresponding ratio was found to be 5000 and for the incisors 
(inclusive of growth) 2700. We also determined the activity of the acid 
soluble phosphorus extracted from the muscles of the rat and found 
1 mgm to contain 0.042% of the labelled P given. From this figure and 
those found for the activity of the tooth and tibia phosphorus 1o bo 
seen in Table 16 it follows that a comparatively fast phosphorus exchange 
is taking place in the muscle compared with that ascertained in Ihe 
l)ones and the teeth. 



EXCH.\N(iE OF PHOSPHOllUS IN' TEKTIL 



183 



EXCHANGE OF PHOSPHORUS IN THE TEETH OF CATS 



"N! 



For the teeth of young cals^ killed a few hours afler Ihc subcutaneous 
injection of the labelled phosphorus the results seen in Tabl(> 17 and 
18 Avere obtained. 



T.A.Br.K 17. Cat VVkichi ntj 2 kgu Killkd aktkh 31/2 Hours 



Tooth 


Weight of iisli 
in ragm 


",j of injoctpd 

laljelleii I' [iresent 

in the tooth 


";,of thchilii'llc.i 

P per mgm ash 

o£ the tooth 


Upper molar 

Lower molar 


123 
110 

108 
91 


O.OK) 
0.014 
0.044 
0.040 


0.00013 
0.00013 


Upper canine 

Lower canine 


0.00041 
0.00044 



Table 18. Cat Weighing 2.5 kgm., Killed after U/j Hoitr.s 



Tooth 


Weiglit of ash 
mgm 


% of iujecteil 

labelled P pa'esent 

in the tooth 


% of tlie liibelled 

P per mgm ash 

of the tooth 


10 Incisors 


94. S 
148 
126.3 


0.0032 
0.019 


0.000034 


Canine 

Jaw 


0.00013 
0.00037 



We also investigated fully grown cats. A cat weighing about 4.5 kgm 
and killed three days after administration of the labelled P gave the 
figures seen in Table 19. In this experiment the labelled P injected was 
not of negligible weight but amounted to 15 mgm (corresponding to 
about 75 mgm sodium phosphate). The labelled phosphorus used in 
this experiment was kindly presented to us by Prof. Lawrence and 
was prepared by the action of high speed deuterium ions on phosphorus 
and accordingly contained a comparatively large amount of normal 
phosphorus. The injection of 15 mgm P into a cat leads to an 
accelerated excretion and the figures are thus not entirely comparable 
with those of the last described experiment, which was furthermore 
carried out on a growing cat. 



^ The heads of the eats were kindly given to us by Professor Lundso.a.aj{I): 
they were obtained in the course of an investigation on the distribution of labelled 
phosphorus carried out by him and one of the present writers. In the first men- 
tioned case 1 mgm plasma P was found to contain after Si/o horn's 1.0% of the 
activity injected, i. e. about 2300 times as much as that present in 1 mgm of tlu> 
upper molar P. 



184 



ADVEXTIKES IX RADIOISOTOPE RESEAECH 



Tahlk lit. Cat Wkighing 4.5 kgm. 


Killed after 3 Days. 




Weight of ash 
iu mgm 


% of injected 

labelled P present 

in the teeth 


% of the labelled 
P per 100 mgm 
ash of the tooth 


Molars 

Upper canine,s 

Lower canines 


690.5 
768.4 
635.2 


0.0080 
0.0076 
0.0068 


0.0012 
0.0010 
0.0013 



The corresponding enamels weighed 29.3, 34.3 and 55 mgm. The 
canine enamel was found to contain less than V'^q of the labelled P con- 
tent of the corresponding dentine. 

In another experiment a strong preparation was administered in 
three portions, 5 days, 2 days and 1 day before killing the animal, each 
portion containing 40 mgm P. The results are seen in Table 20. 



Table 20. Cat Weighing 4 kgm.. Killed after 5 Days. 



Weight of 

teeth 
in mgm 



Weight of 

ash 
in mgm 



% of injected 

labelled P present 

in the teeth 



% of the labelled 
P per 100 mgm 
ash of the tooth 



Molar 323.3 [ 186.0 

Canine [ 422.2 274.3 

8 Incisors 172.5 1 117.5 



0.0027 
0.0038 
0.0021 



0.0015 
0.0014 
0.0018 



The enamel obtained is discussed on page 183. In investigating the 
incisors of rats we I'ound the activity to be due almost exclusively to 
the phosphate of the mineral constituents, the pulp being only slightly 
active. In the earlier experiments conditions were however very diffe- 
rent from those obtaining in the above mentioned case. The uptake of 
labelled P in the teeth of a cat is much smaller than in the incisors 
of a rat and correspondingly the ratio of labelled P in the plasma to 
labelled P in the teeth is much larger in the case of the cat. Now a high 
blood activity will lead to a comparatively high pulp activity and we 
must expect a greater share of the pulp^ in the total activity of the tooth 
in the case of cat teeth. To test this point we removed the pulp of some 
of the canine teeth and compared the activity of the dissected and the 
total canine. We found an activity ratio of 3 : 4, showing that a quarter 
of the activity of the canines of a fully grown cat is due to the 
pulp. 

A comparison of the figures of Tables 17 and 18 with those of 19 and 
20 shows that the uptake of labelled P in young animals is greater than 



1 Human tooth pulp was found by H. C. Hodge, Proc Soc. Exp. Biol. Med. 
35, 53 (1936) to contain 0.70% phospolipins besides other phosphorus compounds. 



EXCHANCiK <)1' I'JtOSI'HOins IX TKKTH 1^5 

in fully grown ones and also thai while in the i'ormer case the canines 
lake up 3 1() 4 times as much labelled P (per mgm ash) as the molars, 
in the latter case no such difference is found. As has already been men- 
tioned above the figures for the two sets of experiments are not entirely 
comparable, but no objection can be raised against a comparison of the 
ratio of the canine and molar uptake, which differs very markedly in 
the case of growing rats from the ratio for fully grown animals. The 
following is a possible explanation of this difference: the lal)elled P 
uptake in the teeth of young rats is due partly to a growth of the teeth 
and not to an exchange process; since in the cat the canines grow faster 
than the molars the uptake is greater in the former case. One would 
be inclined to object to this explanation in view of the short dura- 
tion of the experiment, as the growth in the course of few hours may 
l)e entirely negligible. This objection is however unwarranted. The 
molars of the growing cat weighed 116 mgm and those of the fully 
grown animal 691 mgm. It does not take longer than a few years for the 
growing cat to become fully grown so the yearly growth of a molar will 
be above 100 mgm. 

Let us now calculate the amount of tooth ash formed on the 
assumption that the labelled phosphorus found in the tooth is 
due to growth. A molar of the growing cat took up 0.016% labelled 
P during 3.5 hours. The labelled P which we injected into growing cats 
had in most cases a negligible weight originally, but very soon after 
the injection it mixed with the inorganic phosphate of the plasma 
(corresponding to about 5 mgm P) and from that moment we must 
consider the labelled P as having a weight of about 5 mgm 0.016% 
of the labelled P will therefore correspond to 0.0008 mgm P. The next 
step is that a large part of the labelled phosphorus leaves the plasma 
and is replaced by other phosphorus atoms coming from different bodily 
organs and also from the blood corpuscles. The result is that 0.016% 
of the activity given no longer represents 0.0008 mgm P but a greater 
weight, our scale of indication becoming less and less sensitive. From the 
experiences of Prof. Lundsgaard and one of us on the exchange of 
phosphorus present in the plasma we can estimate roughly that the amount 
of P which corresponds after the lapse of 3.5 hours to 0.016% of activity 
is about 0.008 mgm in the case discussed. To transform from phosphorus 
weight to ash weight we have to multiply by six. The weight of the 
tooth thus increases by 0.04 mgm in 3.5 hours and about 100 mgm 
in a year. The order of magnitude of the growth observed and that 
calculated on the assumption that the uptake of labelled P is due to 
growth is thus the same. 

A very simple but instructive calculation can be carried out in tiie 
case of a fully grown cat into which as much as 120 mgm labelled P 
was injected. We can calculate how many milligrams of these 120 mgm 



l^(^ ADVEXTURES IX RADIOISOTOPE RESEARCH 

are to be found after the lapse of 5 clays in a single tooth. Making use 
of the figures quoted in Table 20 we find that a canine takes up 0.005 
mgm and a molar 0.003 mgm. 



THE BEHAVIOUR OF THE ENAMEL 

The difference in the mechanical properties of dentine and enamel 
is very pronounced. The hardness of anterior enamel is nearly half as 
(Treat as that of hardened toolsteel, while dentine compares closely 
with brassi. The hardness is taken as the pressure in kilograms necessary 
to push a steel ball into the test piece. 

The above mentioned difference is not due to a pronounced difference 
in the relative abundance of the mineral constituents of dentine and 
enamel, as discussed on p. 5, but to the following conditions. The amount 
of organic constituents +w^ater found in dentine is about six times as 
large as the amount present in enamel, the calcification of the enamel 
tissue being thus carried through much more effectively than that of 
the dentine tissue. Bowes and Murray^ found organic matter in human 
enamel to an extent of only 1%. As there is more organic matter^ in 
enamel near the junction with the underlying tissue, the dentine, than 
in the part equidistant from the dentine and the surface of the teeth, 
the outer part of enamel must contain even less than 1% organic matter. 
The latter appears to be* a protein containing tyrosin and resembling 
reticulin. 

Another outstanding difference between dentine and enamel seems 
to be the size and degree of orientation of the crystahites present in 
these. As to the orientation it has been stated^ that enamel of high 
quality gives X-ray diagrams of a high degree of orientation, while 
enamel of poor quality does not. On igniting dentine an X-ray diagram 
characteristic of ^-Ca3(P04)2 is often but not always observed'; this is 
never shown by ignited enamel. As it was found' that i3-Csi^{F0^)^ is 
formed when an excess of PO^-ion is present, it was concluded that the 
dentine apatite often adsorbs an excess of phosphate ion which promotes 
the formation of j3-(\{V0^)^ on ignition. In the case of enamel forming 
larger crystallites, no excess of PO^-ions being present, no j3-(\{FO^).2^ 



1 H. C. Hodge, J. Dent. Res. 15, 251 (193(J). 

2 J. H. Bowes and M. M. Murray, Biochem, J. 29, 721 (1935). 

3 C. F. BoDECKER, J. Dent. Res. 6, 2, 117 (1923). 
* P. PiNCUS, Nature 138, 970 (1936). 

5 J. Thewlis, Naturw. 25, 42 (1937). 

6 W. F. Baxe, M. L. Lefevre and H. C- Hodge, Naturw. 24, 976 (1936). 
" G. Trommel and H. Moller, Z. anorg. Chetn. 206, 227 (1932). 



EXCHANGE OF PHOSPHORUS IX TEETH 187 

formation was observed on ignition. While important inlbrmation imiy 
be obtained by the study of X-ray diagrams the interpretation of the 
latter must l)e made with care. 



PHOSPHORUS EXCHANGE IN THE ENAMEL 

In view of the connection found between tlie content of organic matter 
and phosphorus exchange in the teeth it did not appear very promising 
to look for a pronounced exchange in the enamel. The enamel investig- 
ated by us was in some cases removed mechanically while in others 
we succeeded in separating the enamel of cat teeth after igniting the 
tooth very carefully. The enamel, having a different expansion coeffi- 
cient from the dentine, splits off during the ignition process and 
can thus be removed. The method of separation used recently by 
various workers^, in which the tooth is pulverized and placed in 
an organic liquid of suitable density when the heavier enamel settles 
to the bottom of the tube, is not suitable for our purpose. The 
reason is that some dentine often sticks on the pulverized enamel; 
assuming that the dentine is strongly active and the enamel not, we 
see that the presence of traces of dentine in the enamel might falsify 
the analysis. 

We made several experiments with the enamel of cat teeth but in 
most cases with negative results, the exchange in equal weights of enamel 
being at least 20 times as small as that found in the molars of cats. 
In one case we got a positive effect, the canine of a fully grown cat 
five days after injecting the labelled phosphorus showing a radioactivity 
of 26 relative units (counts per minute), one enamel sample showing 
0.6, and another 0.7 counts. The first mentioned enamel was separated 
by grinding it off from the dentine, while the second one was obtained 
by the same method from the uppermost enamel layer. The ash weight 
of the canine was 277.3 and that of the enamel samples 33.1 and 19.1 
mgm. We are however reluctant to accept this positive result. On account 
of its smaller weight and greater distance from the underlying dentine, 
the outermost layer should be less active than the second enamel 
layer, unless the labelled phosphorus present in the saliva (which 
13.4 mgm. % P 100 cc.) can interact with the outer layer of the 
<'namel. We intend to follow up the problem of the phospho- 
rus exchange in enamel using phosphorus preparations of greater 
activity. 



^ Comp. P. J. Brekhus and W. O. Arm-strong, J. Dent. Res. 15, 23 (1935). 
1 M. Karshan, J. Dent. Res. 15, 388 (1936). 



188 



ADVEXrrRES IX RADIOISOTOPE RESEARCH 



EXCHANGE OF PHOSPHORUS IN HUMAN TEETH 

Other things being equal the exchange of phosphorus in teeth will 
be determined })y the efficiency of lymph circulation in the tooth. 
Exchange experiments can thus be carried out to obtain information 
on the latter point. It does not look improbable that the growth of caries 
will be facilitated by a poor circulation; to decide this point we compared 
the phosphorus exchange in two teeth of the same individual (16 years 
old) removed simultaneously, one on account of caries, the other, a 
healthy one, to space the patients teeth better; about a two hundred 
thousandth part of the labelled phosphorus was found in each of the 
teeth investigated, a quantity sufficient to be measured but not large 
enough to permit the exact comparison necessary to decide the point 
discussed above. The weights of the whole fresh teeth were 800 and 
540 mgm and of the ash obtained on ignition 465 and 330 mgm 
this corresponds to a loss on ignition of 58 and 61%. The time 
which elapsed between the injection of the radioactive phosphorus 
and the extraction of the teeth was 7 days. Through the very great 
kindness of Professor Lawrence we were able to continue these 
experiments using a much stronger radioactive phosphorus sample 

Table 21. 
Labelled Phosphorus in the Teeth of a 25 Year Old Patient 

a) Necrotic Roots. 



Fresh weight 



Asli weight 



Relative labelled P content 



In total 
root 



In 100 mgm 
root ash 



1 

2 

3 

4 
5 
6 
7 
8 
9 
10 



223.7 


138.1 


2.7 


284.1 


190.1 


4.9 


199.4 


127.1 


2.5 ; 


230.5 


143.0 


1.4 


124.8 


76.5 


3.9 


435.2 


268.5 


5.9 


205.7 


127.1 


4.9 


! 169.5 


109 


1.6 1 


172.5 


106.6 


3.8 


183.5 


115.0 


2.1 ; 

i 



1.96 
2.72 
1.97 
0.98 
5.13 
2.19 
3.86 
1.47 
3.57 
1.83 



prepared by him with the aid of his powerful cyclotron. 900 mgm 
labelled sodium phosphate per os w^ere administered to a patient 25 
years old. 4 days later 10 necrotic teeth and 5 days later still, three 
more, fairly well preserved, living teeth were extracted. Of the 2.5 • 10** 



EXCHANGE OF PHOSPHORt'S IN TEETH 



189 



relative radioactive units we can estimate that about 1.8 -lO^ were 
absorbed. As is seen in Table 21/6 relative units were found in a fairly 
well preserved tooth on an average, showing that about 1 : 300.000 
parf of the labelled phosphorus atoms enter a single tooth; in the case 



b) Necrotic C r o w n s 





Fresh weij^ht 


Ash weight 


Relative labelled P content 




In total 
crown 


In 100 mgm 
crown ash 


Onf» sinclp orown 


65.8 
241.7 


39.1 
149.8 


3.7 
12.3 


9.4 


Fragments of several crowns 


8.2 



c) Almost normal roots 





Fresh weight 


Ash weight 


Relative labelled P content 


Xr. 


In total 
root 


In 100 mgm 
root ash 


1 




377.4 
651.1 
685.9 


241.0 
413.6 
430.2 


2.8 
3.7 
6.7 


1.16 
0.9 


3 


1.56 



d) Almost normal crowns 





Fresh weight 


Ash weight 


Relative labelled P content 


Xr. 


In total 
crown 


Into mgm 
crown ash 


1 

2 


533.5 

.S62.9 
464.1 


338.7 

670.9 
429.9 


1.9 

1.8 

1.8 


0.56 
0.27 


3 


0.42 



of a 16 years old boy about 1 : 200 000 was found. In the latter case 
an activity of only 0.5 units (counts per minute) was shown by a 
single tooth, and the estimate was accordingly only a very rough 
one. From the above result W follows that about 1 : 300,000 part 
of the phosphorus taken up with lli(> foot! finds its way into each tooth 
of an adult. 



190 ADVEXTURES IX RADIOISOTOPE RESEARCH 

Summary 

It has been shown that an exchange of phosphorus atoms present in the teeth 
with those present in the blood plasma takes place. 

During the growth of the incisors of rats the newly deposited phosphorus 
atoms are to a large extent found in close vicinity of the dental pulp, but even in 
the most remote part of the incisor presence of newly substituted phosphorus 
atoms can be established. An exchange of phosphorus atoms thus takes place 
even in those parts of the incisors which are entirely outside the range of the 
pulp. The exchange in the molars was found to be less pronunced than that 
in the incisors, this being presumably due to the fact that these do not grow. 

In the teeth of young cats within fews hours., Vjesides an exchange of phos- 
phorus atoms, an increase in the labelled phosphorus content due to the growth 
of the teeth could already be ascertained. 

An exchange of phosphorus has also been proved for human teeth, 1 : 300,000 
of the phosphorus administered being found in each tooth. The replacement of 
1 "o of f^6 phosphorus content of a human tooth by phosphorus atoms taken 
up with the food takes about 250 days. 



Originally puhlishod in Biochcm. J. 34, 532 (1940) 



21. RATE OF REJUVENATION OF THE SKELETON 

G. CH. Hevesy, II. B. Levi and O. II. Rebbe 
From the Institute of Theoretical Physics, University of Copenhagen 

The first experiments in which labelled (radioactive) phosphorus ^-P 
was applied as an indicator [Chievitz and Hevesy, 1935], showed 1 
ihat some of the phosphorus atoms of the mineral constituents of tlu^ 
hone exchange rapidly with Ihose present in the plasma. This result 
was corroborated and extended by later work on this subject [Hevesy 
et ah, 1937; Cook et al., 1937; Dols et al, 1937; 1939; Artom et al., 
1938; (John and Greenberg, 1939; I^efevre and Bale, 1939]. The 
question as to what extent the P contents of the mineral constituents 
of the bone are replaced in a given time by plasma P remained unan- 
swered however. This is an important question, as the rate of this repla- 
cement is a measure of the rate of rejuvenation of bone tissue. The extent 
to which bone P is replaced by plasma P in the course of a given time 
can be determined by comparing the activity of 1 mgm of bone P with 
that of 1 mgm of inorganic plasma P. The activity of the plasma P, i.e. 
its 32P content, changes appreciably, however, with time. The ^-P atoms, 
like all P atoms present at any moment in the plasma, exchange with 
the P atoms present in the various organs and in doing so are removed 
from the plasma and replaced by tissue P. The application of the above- 
mentioned consideration implies a constancy of the activity of the plasma 
inorganic P. To secure such a constancy, we administered labelled phos- 
phate all through the experiment, instead of doing so at the start of th(> 
experiment, as in all investigations mentioned above. By taking blood 
samples at intervals, we ascertained that the activity of the inorganic 
P of the plasma remained constant. At the end of the experiment, the 
l^one sample Avas purified from all non-mineral constituents and Ili<' 
radioactivity of 1 mgm of bone P compared with that ol" 1 mgm ol 
plasma inorganic P. 

EXPERIMENTS WITH FROGS 

Experiments of 5 to 240 min duration wore carried out with frogs. As early 
as 5 min after injecting 0.3 ml. physiological NaCl solution containing a negli- 
gible proportion of labolkvl sodium phosphate into llio lymph sac, the minersil 



192 



ADVENTURES IN RADIOISOTOPE RESEARCH 



Table 1. Labelled P Contents 
OF Plasma and Tibia of a Frog 

Wt. 45 gm: temperature 22°; 

Determinations o min after the start 

of the experiment 



Friiclion 



^^1' content per 

mgm P (specific 

activity) 



Plasma ' 100 

Epiphysis | 0.020 

Diaphysis 0.013 



Epiphysis 




Fig. 1. Extent of replacement of frog's bone P by 

labelled P 



<'onstituents of the tibia contained some labelled phosphate, as shown in 
Table 1. 

With increasing time, the ^-P content of the mineral constituents of the tibia 
increases (Fig. 1). .After the lapse of 1 hr., the specific activity- of the epiphysis 
P amounts onh- to 1/600 of the corresponding magnitude of the inorganic P of the 
j)lasma. Thus only l/tiOO or less of the epiphysial P was replaced by plasma P within 
1 hr. In the next 3 hr. a further 1/900 part of the epiphysis P exchanged. The 
first point in the curve was obtained by anal\sing the right, the second point b>- 
analysing the left tibia. For the diaphysis, the corresponding figures were found 
to be 1/900 and 1/1200, respectively. In the course of 4 hr., therefore, only a minute 
part of the tibia P is replaced by plasma P. A still smaller replacement is fovuid 
when the frog is kept at 0°. 



KATE OF REJUVENATION OF THE SKELETON 



i9:i 



EXPERIMENTS WITH RABBITS 

After the lapse of 2 hr., 1/530 and 1/1800, respectively, of lh(> tibia epiphysis 
P and diaphysis P were found to be replaced by plasma P. With incroiising tinie, 
[\n increase of the replacemont of the l)onc P takes place, as shown in Fig. 2 anrl 
Table 2; this increase diminishes, however, with inc^reasing lime, as would i)c 
excepted. The bone tissue contains numerous small crystals formed by mineraliza- 
tion of the matrix. The crystals are built uj) on similar lines to the mineral apa- 
tite. ^ While the atoms situated on the uppc^-rmost layer of the crystals [Pa- 
neth. 1922] exchange easily with those present in the surrounding liquid, those 
situated inside the crystal are prevented from doing so, except at very high tem- 
peratures. The exchange between bone phosphate and plasma phosphate which 
we observe in experiments of short duration is due to a replacement process bet- 
ween the phosphate ions situated on the surface of the apatite crystals and those 
of the plasma or lymph. Should the surface exchange be exhausted, an increase 
of the time of the experiment may at first have no effect at all on the 
extent of replacement. If, however, in the course of time a dissolution and lep- 
recipitation of the apatite crystals takes place, new and far-reaching possibilities 

Table 2. Extent of Replacement of the Bone P 
OF THE Rabbit by Labelled P 

The labelled phosphate was injected intravenously 
(luring the experiment 



Fraction 


2 hr. % 


4 hr. % 


Epiphysis 


0.180 
0.056 


0.200 


Diaphy.sis 


0.106 




10 



20 30 

dcys 



5C 



Fig. 2. Extent of replacement of rabbit's bone P by hi belled P 



'From X-ray mea-fiirempnt, it was concluded (Caglioti. 193(51 that the inorganic part of the lioiie 
lias the composition of about 3Ca (l'04)2CjiC03j;H20 with the liejcasonal structure of hydroxyapatite, 
the length of the axes being a = 9i"2. < \0^* cm. and <■ ^ 6-9 x 10^* cm, the axis beina oriented parallel 
to the length of the bone. The organic parts consist of polypeptide chains, supported and stretched. 



1 3 Hevesv 



1 94 ADVENTURES IN RADIOISOTOPE RESEARCH 

of an exchange between plasma P and bone P will arise. From these considera- 
tions it follows that an exchange taking place within a long interval cannot be 
extrapolated from results obtained in experiments of short duration. We have, 
therefore, carried out experiments in which we kept the activity of the plasma 
inorganic^ P of rabbits at a constant level for several davs or weeks. 



EXPERIMENT OF LONG DURATION 

To obtain a constant level of the plasma inorganic P, the first day every 30 min 
and later twice everj? day, labelled sodium phosphate of neghgible weight was 
administered by subcutaneous injection to rabbits. After removal of the marrow, 
the bone was first extracted for 12 hr. with hot ether-alcohol. The bone was tlaen 
treated with hot alkaline glycerol solution for further 6 hr. The fractions obtained 
were dissolved in HNO3 and their P contents precipitated as molybdate. The 
molybdate was dissolved in dilute NH3 and precipitated as ammonium magnesium 
salt. An aliquot of the sample obtained was used in the colorimotric P determina- 
tion, while another aliquot was reprecipitated as ammonium magnesium phosphat(> 
and its radioactivity measured with a Geiger counter. In prolonged experiments 
the analysis of the plasma inorganic P is conveniently replaced by that of thc^ 
urine P. In Tables 3 and 4 the specific activities of the different bone P fractions 
are recorded. 

Table 3. Extent of Reji'venation 
OF THE Tibia in the Course of 9 Days 

Wt. of rabbit: 2 kgm 



Fraction 



% P rejuvenated 
I (specific activity) 



Epiphysis P 11.2 

Diaphysis P 3.2 

Tibia phosphatide P . . . 74.8 

Marrow phosphatide P . 1 80.1 



In the course of 9 days therefore, only ll°o of ^he epiphysis and 3% of the 
diaphysis are rejuvenated, while most of the phosphatide molecules present in the 
marrow and in the bone are newly formed. 

In the course of 50 days, only 29% of the epiphysial and 1% of the diaphysial 
mineral constituents were replaced (Table 4). The tibia and femur show about the 
same behaviour. About half of the scapula remained unchanged. The almost 
complete replacement of the apical and medial parts of the incisor dentine P can 
hardly be interpreted as due to an exchange between dentine P and plasma P. 
since the replacement rate of the dentine P was found to be lower than that of the 
tibia P [Hevesy et al., 1937: Lefevre and Baxe, 1939] and since the tibia P, 
as seen above, was replaced only to a restricted extent. The high ^sp content of 
the incisor dentine must bo duo to an actual growth, to a formation by a calcifi- 
cation process. As a plasma containing ^-P was instrumental in calcifying the 
newly grown parts of the incisor, the P of the latter was bound to have the same 
specific activity as shown by the plasma P. As seen in Table 4, the P of the apical 
part of the incisor dentine investigated has, within the errors of experiment ( + 5%). 



BATE OK REJUVENATION OF THE SKELETON 



195 



Table 4. Extent of Rejuvenation of the Skeleton of 
A Rabbit in thk CoriiBE of 50 Days 



Fmclioii 



% rrcjuvenutcd 



Femur epij)hysis inorganic P 

Femnr epiphysis phosphatide P . . . . 
Femur epiphysis glycerol extracl* P . . 

Femur diaphysis inorganic P 

Femur diaphxsis phosphatide P . . . . 
Femur diaphysis glycerol extract* P 

Tibia epiphysis inorganic P 

TiV)ia diaphysis inorganic P 

Costa 

Scapula 

Incisor dentine, apical 

Incisor dentine, medial 

Incisor dentine, incisal 

Incisor enamel, apical -f- medial . . . . 
Incisor enamel, incisal 



29.7 
10(1 

.Jl.O 
().7 
100 

H4:.r, 
28.6 

7.0 
27.5 
43.8 
103 
!»S..-) 
41.2 
.S2.0 

(i.6 



* This fraction presumably contains some mineral P. 

he same specific activity as that of the plasma P. This part, having a length of 
about 0.9 nun., is entirely newly formed during the experiment. The bulk of the 
medial part of the dentine was freshly grown as well, while the incisal part, having 
a length of 1.2 mm., is only partially newly formed with participation of the 
labelled plasma. About half of the P atoms present in the incisal part of the 
dentine were not labelled; they must thus be those which were located in the 
apical or medial region of the incisor before the start of the experiment. The tissue 
containing these atoms was pushed forward m ioto. Partly before this "slipping" 
process and partly during it, some of the P atoms of the dentine have the oppoi- 
tunity to exchange with labelled P atoms and, therefore, the P of the incisal 
part of the dentine shows an activity which amounts to about 1/3 of the specific 
activity of the plasma P. We see here an interesting case of tissue formation in 
which macroscopic aggregates aie "slipped" from one place to another in toto, 
experiencing only a restricted atomic or molecular replacement. This effect is 
much more clearly shown in the growth of the enamel. 

The apical and medial parts of the enamel are formed by a calcification process 
from labelled plasma and, therefore, these parts of the enamel became strongly 
active. From the fact that the incisal part of the enamel is only slightly acitive, 
we have to conclude that this fraction is not formed in the course of the experi- 
ment through a calcification process. Its crystals were formed at an earlier date 
from non-labelled plasma and the whole fraction "slipped" in toto during the 
course of the experiment from the position in which it was caltMfied into the 
place it took up at the end of the experiment. The incisal end of the dentine is 
probably to a large extent also formed by "shp" in toto of the medial parts, though 
this conclusion is not supported as clearly by the activity figures arrived at in 
the case of the enamel. A part of the incisal dentine P bad an opportunity to 
exchange to an appreciable extent before the "slip" took place and also duiing 
that process. Enamel P exchanges only to a minute extent [Armstrong, 1940]. 
It is also of interest to note that the activity figures exclude tlio possibility that 



13* 



196 ADVENTURES IN RADIOISOTOPE RESEARCH 



the incisal part ol the enamel is formed by extensive calcification of the outer 
region of the dentine. In that case, the enamel could not be much less active 
than the corresponding dentine part. The foundation of the incisal enamel is laid 
in the apical end and reaches its final position without interchange with the 
dentine. As both the P and Ca of the teeth have their origin in the plasma, the 
application of labelled Ca as an indicator can be expected to lead to similar results 
to those found above. 



EPIPHYSIAL AND DIAPHYSIAL BONE TISSUES 

The epiphysis was found to exchange the P content of its mineral constituents 
at a higher rate than the diaphysis, as seen in Table 5. 

Table 5. Ratio of the Specific 

Activities of the Epiphysial and 

Diaphysial P of the Tibia of Rabbits 

Tlie level of the activity of the plasma 

inorganic P was kept constant 

all through the experiment 



Time 


Ratio 


2 hr 


3.2 


4 , 


1.9 


10 davs 


3.5 


50 , 


3.S 







In experiments with frogs, in which the labelled phosphate was injected into 
the lymph sac at the start of experiments, taking 1 — 22 days, the ratio 1.3 — 1.6 was 
found. In these experiments, the tibia and femur were both investigated and the 
average was taken. In an investigation of the tibia P of rats, to which the labelled 
phosphate was administered at the start of the experiment, the ratios 3.1, 2.9, 2.5, 
1.7 and 1.8 were found after ~ hr., 4, 10, 50 and 110 days, respectiveh'. It is 
of interest to remark that from the finding that the diaphysial P is only about 
half as active as the epiphysial P 110 days after the start of the experiment, we 
can conclude that an appreciable part of the skeleton has not been renewed 
within 110 days. 

In experiments on chickens, Dols et al. [1939] found the above ratio to be 3, 
22 hr. after administration of labelled phosphate: rachitic chickens gaxc the 
ratio 2.5. 

The more rapid exchange of the epiphysial P is just what would be expected. 
The epiphysis is characterized by a poorer mineralization of the matrix than is 
the diaphysis and contains more organic matter and water than the diaphysis. 
The circulating lymph, containing the labelled P, will therefore reach the apatite 
surface more easily in the first-mentioned case. Should the size of the apatite 
crystals be smaller in the epiphysis than in the diaphysis and theicfore the ratio 
surface: volume Vjc larger in this type of bone tissue, one would also expect a 
more rapid exchange of the mineral constituents of the epiphysis. Whethei such 
a difference in the size of the apatite crystals actuaUy occurs is not known. X-ra> 
investigations [Bale ct al. 1934] lead to the result that the size of the ultimate 



KATE OF KEjrVENATION OK THE SKELETOX 19" 

fuystals ol' (lentinr and bono is ubout 10 " cm.; while the very efi'ectively mi- 
neralized enamel contains larger crystals (10~^ cm.). It is, therefore, quite pro- 
bable that the difference in the extents of mineralization of the epiphysis and 
diaphysis manifests itself in a moderate difference in the sizes of the ultimate 
crystals in the two types of bone tissue. It is of interest to note that the diffoience 
in hardness of the different types of bones is, to a laige extent, the result of a 
different degree of orientation of the crystallites of the bone. X-ray patterns in- 
dicate that orientation occurs during growth and first in those bones where the 
need for solidity (e.g. leg bones) is greatest [Caglioti and Gigante, ]93()]. 



summary 

Labelled (radioactive) phosphate was administered to rabbits and frogs lepeal- 
edly during the experiment in order to keep the radioactive plasma inoiganic 
phosphorus at a constant activity level. The comparison of the activity of 1 mgni 
bone inorganic P with that of the plasma inorganic P permits us to conclude to 
what extent the mineral constituents of the bone were renewed during the expe- 
riment. 

Within oU da_\s, 30% of the femur and tibia epiphysis weie found to be renewed, 
while the corresponding figure for the diaphysis amounted to 7%. Half 
of the mineral constituents of the scapula were found to be unchanged. The phos- 
phatides of the bones and the marrow were entirely renewed. 

The phosphorus of the apical part of the dentine of the labbit's incisor was 
found to have the same activity as the plasma phosphorus. From this result il 
follows that this part of the dentine was grown with the participation of labelled 
plasma phosphorus during the experiment. The greater part of the incisal end of 
the dentine was not formed by a calcification process in situ but by a '\slip" of 
the apical part of the dentine. 

The same behaviour is shown even more pronouncedly by the enamel. No 
interaction of any significance takes place between the incisal part of the enamel 
and the dentine either during their formation or at a later date. The atoms pre- 
sent in the former are to a veiy large extent those which weie proA'iously located 
in the apical (medial) part of the enamel. 



References 

Armstrong (1940) J. hiol. Chein. (in the Press). 

Artom, Sarzana and Segre (1938) Arch. int. Physiol. 47, 245. 

Bale, Hodge and Warren (1934) Amer. J. Roentgenol. 32, 369. 

Caglioti (1936). Atti Congr. naz. Chim. pura appl. 1, 320. 

Caglioti and Gigante (1936) B. C. Accad. Lincei, Classe sci. /is. 23, 878. 

Chievitz and He\t}sy (1935) Nature, Lond., 136, 754. 

Chievitz and Hevesy (1937) Kgl. Danske Vidensk. Selsk. Biol. Medd. 13, 9. 

COHN and Greenberg (1939) J. hiol. Chetn. 128, 116 and 130, 625. 

Cook, Scott and Abelson (1937) Froc. nat. Acad. Sci. 23, 528. 

DoLS and. Jansen (1937) Froc. Acad. Sci. Amst. 40, 3. 

Does, Jansen, Sizoo and Van der AIaas (1939) Froc. Acad. Sci. Amst. 42, 2. 

Hevesy, Holst and Krogh (1937) Kgl. Danske Vidensk. Seltk. Biol. Medd. 

13, 1. 
Lefevre and Baee (1939) ./. biol. Chem. 129, 125. 
Paneth (1922) Z. Elcktrochcm. 28, 113. 



Origimilly published in the Svedberg p. 450. Uppsala (1944) 

22. RETENTION OF ATOMS OF MATERNAL ORIGIN IN 
THE ADULT WHITE MOUSE 

By G. Hevesy 

From the Institute of Theoretical Physics, University of Copenhagen and the Badiiini 

Station in Copenhagen 

What percentage of the atoms present in the new-born organism is 
retained during the later phases of life and what percentage is inherited 
by the subsequent generations? An attempt was made to answer 
these questions by following the fate of the phosphorus atoms in the 
white mouse, radio-phosphorus being used as an indicator. 

The phosphorus atoms present as constituents of different compounds 
in the body of the new-born animal are released successively from the 
compounds in which they are present. The phosphorus atoms thus 
released are either excreted or re-incorporated into various compounds 
present in the body, the latter process being much more frequent. 
Although, for the sake of simplicity, we speak of phosphorus atoms, 
practically no phosphorus atoms but only phosphate radicals are released 
from and built into such phosphorus compounds. The organism is sup- 
plied with phosphorus in the form of phosphate radicals and the phos- 
phorus atoms adhere, as far as is known, to their partners throughout 
the numerous metabolic processes in which they participate. 

The white mice used in the experiment were kept on the following 
diet. Wheat flour, oatmeal and a small amount of milk were administered, 
while on alternate days only Cooley's standard food was provided. 
Once a week, cabbage or lettuce was administered as well. 

About 0.1 mgm of labelled sodium phosphate with an activity of a few 
microcuries was administered by subcutaneous injection to a pregnant 
mouse. As a result of introducing radiophosphorus into the organism 
of the pregnant mouse, we obtain offspring of which the phosphorus 
contents were labelled. A litter consisted of about 8 offspring having 
almost the same weight, as shown in Table 1. 

The radio-phosphorus contents of the offspring may therefore also 
be expected to be almost equal. This fact makes it possible to determine 
the total ^^P content of the offspring at any date by measuring the 
total activity of any member of the litter. 

One offspring was killed shortly after birth and dissolved in concentra- 
ted nitric acid. The phosphorus content of a known aliquot of the solution 



RETENTIOX OF ATOMS OF MATERNAL ORIGIN IN THE ADtLT WHITE MOT'SE 199 

was precipitated as magnesium ammonium phosphate. The precipitate 
was filtered through an aluminium dish oi" 1.1 cm diameter having a 
perforated bottom covered with filter paper. The dish containing the 
precipitate was tlion plaoo(l unter the (Joiger counter. By comparing 



Table 1. — Wkight of 6 
Nkw-bornt Offsprino of a Motsk 



No. 


Weight in gm 


Relative 
activity 


1 


1.3 


lOU 


2 


1.3 


98..') 


3 


1.1 


99.0 


4 


1.2 


93.5 


5 


1.3 


99..5 


6 


1.3 


— 



the activity of an offspring killed at a given date with the activity of 
another killed at a later date, it was possible to calculate what percentage 
of the phosphorus atoms of maternal origin was lost in the interval 
between the two dates. 

All offspring were killed successively, dissolved, and treated in the 
way described above. The writer is much indebted to Mr. K. Zbrahn 
for dissolving the mice and precipitating their phosphorus content. 
All offspring were killed and investigated within about three months. 
After the lapse of this time, the activity of the phosphate precipitates 
had decreased so greatly that it could no longer be measured with suffi- 
cient accuracy. The activity of the first offspring was compared with 
that of the second, the activity of the second with that of the third, 
and so on. 

The mouse obtains its ^-P content not only by birth but also by 
lactation. In order to simplify the problem, to reduce the ^^P content 
of the offspring mainly to such ^^P as was obtained by birth, the active 
mother was replaced by an inactive mouse soon after gestation. As the 
replacement of the mother was not made immediately after the birth 
of the offspring, we actually measured the loss of ^^p acquired by birth 
plus the ^^P acquired by lactation in the interval between birth and 
replacement of the active mother by an inactive one, i.e. within a ftnv 
days. 



200 



ADVENTURES IX RADIOISOTOPE RESEARCH 



RESULTS 

Tho result obtained are shown in the following tables. 

Table 2. — Mother injected Febrxarv 
9. Date of gestation: February 

18. Replacement of the active by an 

INACTIVE mother: Febri'ary 22. 

(Experi:\iknt 1.) 



No. of 
offspring 


Killed 


Relative 
activity 


Weight 
in gm 


1 


22/2 


lUO 


3 


2 


3/3 


82 


7 


3 


16/3 


73 


ir, 


4 


30/3 


4S 


18 


5 


13/4 


41 


25 


6 


13/5 


40 


23 



,i 'Ji 



Loss of ^'~i' in the course of 81 days: 60 per cent. 

In about 3 months, ^ a time sufficient for mice to reach adulthood. 

Table 3. — Mother injected February 
9. Date of gestation: February 16. 
Replacement of the .active by 
->.^ an inactive mother: february 23. 

(Experiment 3.) 




No. (if 
offspring 


Killed 


Rehitivc 
:iotivity 


Weight 
in gm 


1 


24/2 


100 


2.8 


2 


5/3 


90 


().9 


3 


19/3 


70 


13 


4 


2/4 


57 


15 


5 


16/4 


53 


20 


6 


18/5 


51 


16 


7 


7/6 


39 


22 



Loss of ^"P ill the course of 103 days: 01 per cent. 

only about 60 per cent of the ^^p content of the mouse acquired by birth 
is thus eliminated. The phosphorus content of the new-born mouse is 
found to be about 4 mgm the amount of P excreted by the mouse in the 



1 If only the last two values in Tables 2 and 3 are considered the average 
loss of 32P in 80 days works out at 57 per cent. If all mice killed in Ajaril, 
-May and June are considered, the average loss is 56 per cent in 72 days. 



JJETENTIO.N Ui' ATOMS OF .MATKllNAL OJUUl.N IN TUE AUlLi WHITE .MOUSE 



21)1 



course ol' three nionlh uboul 1 gniThe lad lliat neailx one half of the 
maternal phosphorus is retained in the body, in spite of th(^ 
comparatively large amounts of phosphorus excreted by the mouse 
in the course oi" 3 months, is due mainly to the protection of a large 
proportion of the phosphorus of the bones against interchange with the 
])hosphorus atoms in circulation. The uppermost atomic layers of the 
bone apatite crystals interchange easily with the phosphorus atoms 
of the plasma or the lymph; furthermore, a kind of biological "recrystalli- 
zation" takes place, i.e. dissolution of some molecular apatite layers 
foUow'cd by new formation of such layers through crystallization. .\ll 
these processes, however, do not affect, or affect only at a very slow 
rate, large parts of the bone apatite which thus retain their P atoms. 
During the formation of the skeleton, a large proportion of the ^ap 
atoms present in the organism will find their way into the bone apatite 
and be fixed there to a very appreciable extent fluring the time of the 
experiment (3 months) or even for the lifetime of the mouse. 

When comparing the ^^P content of several rats injected simultaneously 
with labelled phosphate at different dates, it was found (IIevesy 1939) 
that the ^'^P, in so far as it was not excreted, accumulated to a very 
large extent in the skeleton. This fact is illustrated by the following 
ta])le. 

Table 4. — Percentage ^^p Present in the 
Body FoTJxn ix s^omf Oikiaxs of the Rat 



Organ 




Time after which the 


rat was Ivilled 




1/2 hour 


4 hours 


10 days 


20 days 


30 days 


50 days 


98 days 


Muscles 


18.3 

37.0 
19.1 


19.4 

13.6 
23 4 


25.8 

21.0 
13 1 


28. S 

16.4 
13.1 


25.2 

13.1 
51.8 


12.1 

6.7 
76.5 


3.6 


Carcass after removal of bones, 

mtiscle.s, blood and skin 

Bones 


2.9 
92.6 











Furthermore, when comparing the specific activity (activity of 1 
mgm P) of the bone P with the specific activity of the plasma P of the 
rabbit in experiments where the activity of the plasma was kept a1 a 
constant level throughout, it was found (Hevesy et al., 1940 — 2) that 
70 per cent of the epiphysial P and 93 per cent of the diaphysial P of 
the tibia remained unchanged after 50 days. These facts illustrate the 
ability of the skeleton to prevent an interchange of a large part of its 
P atoms with the P atoms of the plasma, thus preventing an ultima1<> 
excretion of such atoms. 

As the calcium atoms of the organism are found to a still higher 
percentage in the skeleton than the phosphorus atoms, the organism 
may be expected to retain the average calcium atom obtained by birth 



202 ADVENTURES IX RADIOISOTOPE RESEARCH 

still more jealously than it does the maternal P atoms. Similar conside- 
rations may apply to the magnesium and fluorine content of the organism. 
The atoms of maternal origin of all other elements present in the organism, 
however, can to be expected to leave the body at a much higher rate 
than do the phosphorus atoms of maternal origin. 



THE PERCENTAGE OF 32p TRANSFERRED FROM ONE GENERATION 

TO ANOTHER 

Some experiments were carried out in order to follow the fate of the 
^^P administered to a mouse in the second and the third generation. 

The phosphorus content^ of the new-born mouse constitutes about 
2.4 per cent of the mother's. phosphorus content; consequently, we should 
expect to find about 2.4 per cent of the ^^p contained in the pregnant 
mother in each new-born mouse. Actually, only about 1/4 of that amount 
is found when comparing the activity of a mouse of the third and the 
second generation. This finding is explained mainly by the fact that 
a large part of the ^-P content of the mother is to be found in the skeleton 
and, as a large part of this ^sp Joes not reach the circulation, it does 
not participate in the formation of the foetus. The foetus acquires its 
phosphorus content mainly from the food phosphorus and the phos- 
phorus present in the soft tissues. The phosphorus present in the circu- 
lation has a much smaller ^ap content than the average phosphorus 
of the mother, a fact which results in a comparatively low ^^p content 
of the new-born mouse. The first generation contains labelled phosphorus 
mainly in the soft parts of the body and in a minor part of the skeleton, 
and this because it obtained its phosphorus content by subcutaneous 
injection and not by the much more intimate foetal processes. The 
32p content of the first generation is therefore not strictly comparable 
with that of the second generation. The ^^p contents of the second and 
the third generations may, however, be compared. We may expect 
this ratio to be equal to that which we would obtain on comparing the 
32P contents of mice of the third and fourth or of the fourth and fifth 
generations etc. since it is to be expected that the percentage of ^ap 
passed from the third to the fourth generation will be the same as that 
transferred from the second to the third generation, etc. This conclusion 
is important since it is almost impossible to follow the fate of ^'-P through 
more than three generations of mice. 

From the injection of labelled phosphate to a mouse in the last stage 
of pregnancy (first generation) to the birth of tho fourth generation 

1 The P content of a new-born mouse weighing 1.33 gm average value amounts 
to 4.2 mgm while that of a movise weighing 31.8 g to 177 mgm 



RETENTION OF ATOMS OF MATERNAL ORIGIN IN THE ADULT WIIITK MOUSE 203 

about 188 days elapse; this corresponds to 13 half- lite periods of radio- 
pliosphorus. During this time, the activity administered to the first 
mouse has decreased to less than 1/8000 of its original value. As the 
lourth generation may be expected to contain about 10~" of the 32p 
present in the first generation (apart from the radioactive decay), we 
may expect to find only about 10~ii of the radio-phosphorus adminis- 
tered to the first generation. The measurement of such a low percentage 
of radio-phosphorus administered would require the administration 
of 100 millicuries ^^p or more. 

The phosphorus content of an adult mouse amounts to about 200 mgm 
()!• to 4- 1021 atoms. As of the ^^p atoms present in a mouse only 14 P^r 
cent are found in a mouse of the next generation, it is easy to show- 
that the eleventh generation will no longer contain a single ^^P atom 
and thus no P atom at all which was present in the first generation. 

This result illustrates strikingly the fact that the hereditary disposi- 
tions are entirely independent of any atomic community with the fore- 
fathers, these dispositions being determined exclusively l)y the faculty 
of the atoms and molecules to enter certain characteristic configurations. 
It is the pattern which matters and not the single brick. 

Summary 

Labelled phosphate was administered to a pregnant mouse. Each offspring 
was found to contain aknost the same amount of ^^^P. By kilhng the various offspring 
at different dates and comparing their ^ap contents, we determined the amount 
of maternal phosphorus atoms present in offspring at different times. 

Between birth and maturity, i.e. in the course of three months, the mouse 
lost about 60 per cent of the P atoms acquired by birth. 

By breeding three generations of mice, to the first generation of which ^^V 
Avas administered, the passage of phosphorus atoms from one generation to the 
next was followed. 0.6 per cent of the ^~P present at the birth of an offspring 
of the second generation was found to be present in an offspring of the third 
generation. We might expect to obtain the same ratio between the ^'-P content 
of the third and fourth generation, and so on. 

Making this assumption it can be shown that a mouse of the eleventh generation 
no longer contains a single phosphorus atom present in the first generation. 

References 

Hevesy G. (1939) J. Chem. Soc. 1213. 

Hevesy G. C., Levi H. B. and Rebbe O. H. (1940) Biochem. J. 34, 532. 



Originally- published in Acta Fhijfiiol. Scand. 9, 234 (1945) 



23. RATE OF RENEWAL OF THE FISH SKELETON 

(J. Hevesy 
From the Kristinebergs Biological Station, Sueden 

The phosphorus atoms present in the organism migrate from molecule 
to molecule and from organ to organ. The rate of migration greatly 
depends upon the nature of the molecules involved and on the organ 
in which they are located, the phosphorus atoms finding their most 
lasting abode in the skeleton. The mineral constituents of the skeleton 
are located in apatitelike crystallites which have a size of about 10"^ cm. 
From the phosphate ions present in these crystallites, only those located 
in the uppermost molecular layer can come into direct contact with 
the lymp or the plasma and, thus, participate in an interchange with 
the phosphate present in the plasma (lymph). The replacement of 
the bulk of the phosphate or other ions present in the apatitelike 
crystals can only take place by a partial or total dissolution of the 
crystallite followed by a crystallization process leading to a partial or total 
formation of new crystallites. This process is made possible by the 
fact that the concentrations of phosphate and of other constituents 
of the plasma vary. Intake of food increases the phosphate content 
of the plasma and the lymph, and so do numerous biochemical pro- 
cesses leading to an enzymic splitting of organic phosphorus compounds. 
The plasma phosphate, for example, increases after intense muscular 
action, though the low phosphate permeability of the muscle cells 
(Hevesy and Rebbe, 1940) much reduces the exodus of the phosphate 
ions split off during muscular action. On the other hand, excretion of 
phosphate acts in the opposite direction, and so do all those numerous 
biochemical processes in the course of which phosphate becomes incor- 
porated with organic^ compounds, from which processes the decrease 
of the phosphate content of the plasma under the action of insulin 
is possibly the most conspicuous one (cf. Kjerulf-Jensen and Lunds- 
GAARD, 1943). 

Not only do the phosphate and the equally important calcium con- 
centrations of the plasma fluctuate, but the same applies to the con- 
centration of phosphatase and other enzymes regulating the phosphorus 
metabolism. Such enzymes act on the bone formation not only by 



RATE OF REXEWAL OF THE FISH SKELETON 205 

regulating the j)lu)sphate concentration of tlie plasma, hut possihly 
also in a more direct way, as suggested by Robison's (1912) earl\ 
studies on bone formation. More recent work by Roche and Mourgue 
(1939) leads to Ihc result thai a fracture of the rat's femur involves 
a loss of appreciable^ amounts of the mineral constituents of the femur 
followed by an opposite process after the lapse of about one month. 
In the first weeks, phosphatase activity of the bone is also enhanced. 
Roche and Mourgue made the very interesting observation that the 
fracture of the left femur leads not only to an initial decrease in the 
mineral content of the fractured left bone, followed later by a reversal 
of this process, but a similar ])ehaviour is also shown by the intact 
right femur. The enchanced phosphatase activity of the bone tissue 
may be due to an increased magnesium concentration produced ])y 
osteolysis following the fracture. Thus, even a fluctuation in the magne- 
sium content of the plasma may promote the rate of renewal of the ske- 
leton. 

Fluctuations in the phosphate, calcium, magnesium, and phosphatase 
contents of the plasma thus make possible a biological recrystallization 
of bone apatite and, corespondingly, a renewal of the skeleton. That 
this process, which can be followed by making use of isotopic indicators, 
was found to be a slow one is easy to understand. The bone apatite 
contains a very appreciable part of the body's calcium and phosphorus 
contents, anfl these constituents are present in a crystalline state. 
Dissolution and formation of such crystallites may be expected to \)v 
a slow process. Furthermore, we must envisage the probability that a 
partial dissolution of a crystallite may be followed by a new formation 
of some molecular layers which protect the underlying part of the crystal 
from further changes. This process can often be repeated and leads to a 
repeated renewal of a fraction of the crystallites, while the remaining 
part of the crystal remains unchanged. The pronounced difference in the 
rate of renewal of epiphysial and diaphysial tissues found by various 
workers is due mainly to the better circulation taking place in the soft 
epiphysial bones but, possibly, to some extent to the smaller size of the 
crystals of the epiphysial tissue which favours an interchange between 
lymph (plasma) and bone phosphate. 

In this paper are communicated the results of experiments carried 
out with the aim of measuring the rate of renewal of the mineral consti- 
tuents of the fish skeleton. However, a short survey of the results 
hitherto obtained for the rate of renewal of the skeleton of mammalia 
will first be given. 



206 ADVENTURES IN RADIOISOTOPE RESEARCH 

RATE OF RENEWAL OF THE SKELETON OF MAMMALIA 

As a result of the administration of labelled phosphate (phosphate 
containing a minute percentage of the radioactive phosphorus isotope 
^^P), the "free" phosphate of the blood plasma soon becomes labelled, 
and the same applies to the extracellular fluid of the organism in view 
of the swift passage of phosphate ions through the wall's capillaries. 
As the plasma and the lymph contain labelled phosphate, all bone apatite 
formed after the administration of labelled phosphate is bound to be 
labelled. In the extreme case, when all mineral bone tissue is formed 
after the administration of labelled phosphate, 1 mgm bone P will have 
the same ^^P content, and thus the same radioactivity, as 1 mgm plasma P. 
Thus, the ratio of the specific activity (activity of 1 mgm) of the skeleton 
P and the specific activity of the plasma P is a measure of the rate of 
renewal of the skeleton. 

When determining the rate of renewal we must take due regard to 
the fact that the specific activity of the plasma phosphorus does not 
remain constant, but decreases strongly in the course of the experiment, 
owing to successive interchanges of the plasma phosphorus with the 
phosphorus atoms of the various compounds present in the organs 
and also to excretion of phosphate. As the rate of interchange is diffe- 
rent for different compounds and also for different organs, the calculation 
of the decrease of the specific activity of the plasma P wath time 
encounters difficulties. The most direct way to eliminate the above 
mentioned difficulty is to administer repeatedly an appropriate amount 
of labelled phosphate, and, with the aid of this procedure, to keep the 
plasma phosphate at a constant level throughout the experiment. The 
results of such experiments (Hevesy et alia 1940) carried out on 
rabbits are seen in Table 1. 

As seen from the figures, the degree of renewal of the mineral consti- 
tuents of the skeleton in the course of 50 days amounts to 30 per cent 

Table 1. Extent of Reneval of the Skeleton of a Rabbit 
IN THE Course of 50 Days 



Fraction 



Per cent 
renewed 



Femur epiphysis inorganic P I 29.7 

Femur diaphysis inorganic P I 6.7 

Tibia epiphysis inorganic P 

Tibia diaphysis inorganic P 

Costa inorganic P 

Scapula inorganic P 



43.8 
Femur epiphysis phosphatide P l 100 



28.6 
7.6 



RATE OF KENKWAL OF THE FISH SKELETON 207 

in 1he case of the ('pi])hysis oi' ihc libia and 1o as lit lie as 7 per cent 
for the diaphysis, while phosphatides extracted from tlie bone tissue 
are entirely, and possil)ly e\-en several times, renewed in the course 
of the experiment. 

The experiments mentioned above were carried out wilh fully grown 
rabbits, as in a growing organism the presence of labelled atoms cannot 
be interpreted exclusively as the result of a renewal process. It will 
also be due to the formation o{ additional tissue duiing the experiment. 
All molecules formed in a growing, labelled organism are, indeed, bound 
to become labelled. It is of importance, therefore, to carry out experi- 
ments on the renewal of the skeleton in adult animals. 

The incorporation of labelled phosphate into mineral components 
of the bone is a very intricate process. Between the uppermost molecular 
layer of the apatite crystallites in contact with plasma or lymph, an 
exchange equilibrium can be established almost immediately. This 
means that the specific activity of the P present in these layers will 
promptly follow all changes in the specific activity of the plasma phos- 
phorus. In most experiments with labelled phosphate, the active prepara- 
tion is administered at the start of the experiment. After subcutaneous 
injection or administration by mouth, an increase in the plasma acti- 
vity will take place in the initial phases of the experiment and a decrease 
throughout the later phases. Thus, the activity of the uppermost layer 
of the bone apatite is strongest in the early phases of the experiment 
in which the plasma is strongly active. Crystallites, how^ever, formed 
in the course of the experiment from an active plasma, will contain 
comparatively large amounts of ^^P in view of the high phosphorus 
content of the total crystallites compared with the phosphorus content 
of the uppermost molecular layer. In view of the stability of the crys- 
tallites, much of their ^^p content will be conserved and will not follow or 
follow^ only slowly the changes in the activity of the plasma phosphorus. 

Beside formation of entire crystallites from the labelled plasma we 
have also to consider the case of partial formation of crystallites. Some 
molecular layers are dissolved and replaced by layers formed by cris- 
tallization from labelled plasma. The newly formed layers will be active, 
but not the layers lying below. These layers will be protected from all 
action of the labelled plasma and, thus, from renewal. They will form 
a stable core for the crystallites and so will all crystallites that are not 
in contact with plasma or lymph. 

Manly and his colleagues (1940), who carried out extensive studies 
into the uptake of ^^p i^y ^^^ mineral constituents of the bone where the 
activity was administered at the start of the experiment, estimate the 
share of labile and stable fractions of the bone tissue by comparing the 
activity of the blood (not of plasma) P and of the bone mineral of rats. 
They estimate Vg of the ^ap content to be present after llic lapse of 20 



908 ADVENTURES IN RADIOISOTOPE RESEARCH 

days in the labile portions of the epiphysis, the rest being found in the 
stable portion. The estimation of such magnitudes encounters great 
difficulties in view of the very complicated way in which the labeUing of 
the bone tissue takes place. The degree of renewal of the mineral consti- 
tuents of the different parts of the skeleton which takes place within 
a time interval can, however, be determined in the way described on 
p. 204. The degree of renewal in these experiments means the percentage 
of bone tissue newly formed once or several times since the start of 
the experiment. 

RATE OF RENEWAL OF THE FISH SKELETON 

We investigated the rate of renewal of the skeleton of sticklebacks (Gasterosteus 
■actileatus). These fish, weighing 1 — 3 gm have a lifetime of about one year and 
can be expected not to grow any longer when one year old. Their small size has 
the advantage that the use of a large sea-water volume and, thus, an unduly 
large amounts of labelled phosphate, can be avoided. Our experiments, in which 
the sticklebacks were kept for up to six weeks in labelled sea-water, were carried 
out with radio-phosphorus having an initial activity of 0.05 millicurie, which 
was found to be ample to give an active skeleton. 

The small size of the fish facilitates, furthermore, their ashmg, which is to be 
carried out when we want to determme the total phosphorus content or the total 
32P content of the fish. Wet ashing was carried out by heating with 1 ml of cone, 
sulphuric acid containing some nitric acid and, in the last phase of the experiment, 
some hydrogen peroxide. 24 sticklebacks were kept in 3 htres of sea- water to 
which 10 ml of a radioactive solution containing 0.05 millicurie and 3.4 mgm of 
sodium phosphate (pH — 7.6) were added. After the lapse of 6 weeks, this acti- 
vity declined to Vs of its initial value. The water was daily renewed, as was its 
labelled phosphate content. 

The sticklebacks were investigated at different intervals. After killing the fish 
and washing it very carefully with sea-water, the liver was taken, and the "free" 
phosphate was extracted from it with cold 5 per cent trichloracetic acid. The solu- 
tion was then brought up to 25 ml. While the "free" phosphate content of 20 ml 
was precipitated as magnesium ammonium phosphate, the activity of which was 
measured, the residual part used in a colorimetric determination of the free P 
content of the extract. The activity measurements are much simplified when 
the samples have about the same weight. To obtain such samples, we added to 
the above mentioned 20 ml so much sodium phosphate that the precipitate obtained 
weighed 60 mgm. As mentioned above, the determination of the rate of renewal 
is hased upon the comparison of the activity of 1 mgm of free plasma P and 1 mgm 
of mineral skeleton P. It is, however, extremely difficult to secure blood from 
fish weighing a couple of grams and, therefore, we replaced the determination 
of the activity of the plasma P by a determination of the activity of the free liver P. 
In view of the great ease with which phosphate ions penetrate the liver cells and 
vice versa, the activitv level of the free phosphate P of the liver differs not much 
from the activitv level of the free phosphate of the plasma. The writer is much 
indebted to Mr." Tryggve Gustavson for his very effective help m removing 
the hvers, weighing 45 — 70 mgm. 

To remove the organic constituents of the skeleton, we treated the bones with 
boiling glycol containing 6 gm KOH per 100 ml for several hours, until the bones 



KATE OF RENEWAL OF THE FISH SKELETON 



209 



showed the total absence of iion-inineial constituents. The bones were dried at 
105° and dissolved in 2 ml 0.5 N HCl. The solution was brought to 25 ml and an 
aliquot was used, as described above, in the radioactive measurements, while 
llie other aliquot was fakcMi for colorimetric determination. 

UPTAKE OF LABELLED PHOSPHATE BY THE FISH 



The uptake of IuIk'HimI phosphate by the fisli most j)n)baljly takes 
place either through the gills or tlirough the digestive tract. While water 
passes the surface of the gills, some phosphate may reach the circulation. 
An increase of the water volume passing the gills may in this case be 
expected to lead to an increase in the phosphate uptake. Therefore, 
we have compared the phosphate uptake by fish kept in water rich 
in oxygen with the uptake of phosphate by fish in water containing 
but very small amounts of oxygen. While a group of fish was kept for 
a day in 2 litres of labelled sea- water saturated throughout the experi- 
ment, another group was kept in 2 litres of labelled sea-water to which 
no oxygen was added and in which other sticklebacks were previously 
kept in order to remove much of its air content. As seen in Tal)U^ 2. 
the average uptake of ^^p l^y the two groups of fish practically does 
not differ. 

Table 2. — Uptake of ^-Pby Fish Kept in Oxy- 
gen-rich AND Oxygen-poor Water in the Course 
OF 24 Hours 



Weiijht of fisli in giu 



Total activity present in 1 gm fish 



water ricli in 
oxygen 



water poor in 
oxygen 



0.82 . . 




20 
15 
25 
21 
17 
24 
16 
21 

20 




2.07 




1.77 




1.53 




1.25 




1.03 

115 




1.30 




0.91 


23 


0.8S 


21 


1.28 


13 


2.63 


19 


1.71 

1.51 

1.24 

1.56 


26 
22 
20 
24 


Mean value . . 


22 



14 Hevesy 



210 ADVENTURES IN RADIOISOTOPE RESEARCH 

The fact that no significant difference was found in tlie uptakes of 
32p from waters rich and poor in oxygen does not prove conclusively 
that the main uptake of ^^P does not take place through the gills, as 
it is possible that the organism reacts to oxygen shortage in the water 
not by an increase in the water circulation through the gills, but by an 
enhanced oxygen extraction from the water. However, the above 
result induces us to draw our attention to the other probable way of 
entrance, the digestive tract, which, as marine fish drink large quanti- 
ties of the water in which they swim, is the most probable path of ionic 
uptake by such fish. 

Homer Smith (1930, 1931) added phenol red to aquarium water and 
found that the dye became concentrated in the intestine and also that 
it could not be absorbed through the gills and the skin. By measuring 
the concentration of the dye he was able to calculate the extent of water 
absorption taking place^. An eel weighing 143.5 gm, was found to have 
Swallowed in the course of 20 hours 12.3 ml of sea-water. A number of 
experiments on eels and sculpins show that per kgm of weight those 
fish Swallow from 40 to 225 ml of sea-water per day. The minimum 
swallowing observed is thus 0.04 mlpergm per day. The amount of labelled 
phosphorus present in 4.04 ml of water is that found by us in a stickle- 
back weighing 1 gm after the lapse of 16 days. K the amount of water 
swallowed by the stickleback is not still larger than the largest amount 
observed in eels and sculpins, then we have to conclude that only a 
part of phosphate present in the swallowed water is absorbed by 
the sticklebacks. This result is by no means improbable. Though 
water is very easily absorbed, Homer Smith found that only 81 per 
cent of the water swallowed by the eel had been absorbed and, further- 
more, that, while monovalent ions such as Na+, K+, and Cl~ were 
absorbed to a very large extent, divalent ions such as Ca"*""*", Mg"^"*", 
and SO^" were not, these ions being concentrated in the rectal fluid. 
In relation to chlorine, sulphate was, for example, concentrated 24 
times in the rectal fluid, (ef. A. Krogh, 1939). The result obtained by 
us suggests a similar fate of the phosphate ions. 

A fish weighing 0.92 gm. took up in the course of 8 days 4 • 10"^ per 
cent of the activity of the labelled sea-water and, thus, as the phosphorus 
content of the sea- water was 880 y (730 added -f 150 present beforehand), 
3.5 7 of phosphorus were taken up mainly as sodium phosphate by the 
fish. The amount of phosphate taken up by the fish during the same 
time varies appreciably from fish to fish. However, these variations 
do not influence the results obtained for the rate of renewal since, when 
calculating this magnitude, the skeleton activity and the liver activity 
of the same fish are considered. 

1 A dofailod account of the work of Homer Smith is given by Krogh (1939) 



KATE OF RENEWAL OF I'UE FISH SKELETON' 21 1 

Gasterosteus aculcatus is a fish provided with a skin armour^ Tn several 
cases, we investigalcMl Ihe ariiiour'. the cranium, and the vcniebra sepa- 
ralely. 

EXTENT OF RENEWAL OF THE SKELETON 

The method ol' calculating^ the extent of renewal of the skeleton is 
shown by the following example. 

Duration of the experiment = 16 days. Average temperature = 16.6'^. 
Fresh weight of the fish = 1.49 gm. Weight of the fresh liver = 50 mgm. 

Free P content of the liver = 21 y. 

Total P content of the skeleton = 3.98 mgm. 

Activity of the skeleton P = 29.2 counts per minute. 

Activity of the free liver P = 8.8 counts per minute. 

Activity of 1 y skeleton P _ 0.00733 
Activity of 1 y free liver P 0.418 

Activity of 1 }' skeleton P in percentage of the activity of 1 y liver P 1.8. 

The figure obtained is not strictly identical with the percentage of the 
skeleton which, in the course of the experiment, is renew^ed, as we com- 
pared the specific activity of the bone P at the end of the experiment 
with the specific activity of the free liver P at the same date, while 
we should have considered the average value of the specific activity 
of the free liver P prevailing throughout the experiment. This magnitude 
is not known, but cannot be less than V2 of the final value. Therefore, 
we have to multiply, the result of 1.8 per cent arrived at by a figure 
which is less and probably appreciably less, than 2 in order to arrive 
at a correct renew^al percentage which, thus, amounts to 3 — 4 per (!ent 
in the course of 16 days. This percentage of the skeleton w^as renewed 
once or several times, while the remaining 96 — 97 per cent of the skeleton 
remained unchanged. 

The fish was kept in 3 litresof sea-water to which 3.4 mgm of labelled 
phosphate, corresponding to 0.73 mgm P and having an activity of 1 .6 • 10<^ 
counts, were added (measured the same day as the activity of the liver 
and that of the skeleton). Water and activity were daily renewed. 

1 y free liver P was found to contain part of the activity and. 

^ 4-106^ 

thus, from the free P extracted from the liver 2-10"'* -/ were sucli 
which originate from the labelled phosphate added to the sea-water. 

The total free liver P contained part of the activitv added to 

1.8-10^ 

^ A detailed de.Sf'i'iption of ihe armour is given by F. Roth (1920). 
14* 



212 



.VDAEXirRES IN RADIOISOTOPE RESEARCH 



the water. The skeleton contained 1/5.3- 10* part of the activity added 
to the water, thus of the 3.98 mgm P present in the skeleton, 1.4- \()-^y 
were such which originate from the labelled phosphate added lo 1 Ik^ 
sea-water. 

The results obtained for different parts of the skeleton are conijni- 
ted in Table 3. 



/ 



Table 3. — Percentage Ratio of the Activitv of 1 -M(;m 
Skeleton P and 1 mgm Free Liver P 



■ 








Percentaffe I'atii) <>l 


No. of 


Fresh weight of 


Time 


Tart of tlie 


tlie activity of 1 mgm 


e\-pc-rinieiit 


the fish in f^iu 


in diiys 


skeleton 


skeleton P and 1 mgm 
liver P 


• 






Skull 


1.7 


19 


1.83 


17 


Vertebrae 
Armour 


1.1 

1.2 


IS 


2.20 


20 


Armour 


2.3 


21 


1.78 


2(t 


Skull 
Armour 

Skull 


2.7 
2.1 

2.4 


22 


1.21 


21 


Vertebrae 
Armour 


3.4 
2.6 


23 


1.33 


30 


Vertebrae 
Armour 


4.1 

2.4 


24 


1.41 


31 


Armour 


4.2 



From the above figures, the average value for the percentage ratio 
of the activity of 1 mgm skeleton P and 1 mgm liver P works out to be 2.5 
per cent in the course of 22 days. To obtain the percentage renewal of 
the fish skeleton in the course of 22 days, we have to multiply the above 
figure (cf. p. 209) by a figure which is less than 2. The rate of renewal 
thus lies between 2.5 and 5 per cent. 



UPTAKE OF LABELLED FHOSPHORLS BY THE EGGS 



In several fish, a large number of eggs was found and in many cases 
the weight of the eggs constituted a very appreciable percentage of the 
weight of the fish. In experiment No. 16 B, the eggs' weight was found 
to be 0.92 gm out of a weight of 2.44 gm of the fish (including eggs), i. e. 
38 per cent. The percentage ratio of the activity of 1 mgm average fish P 
(minus eggs) and 1 mgm free liver P was found to be 6.3. For the percen- 
t age ratio of the activity of 1 mgm egg P and 1 mgm liver P, 90 was obtained . 
Thus, almost all P of the atoms present in the egg was incorporated 
into the eggs in the course of the last 23 days. The total P content of the 



RATE OF RENEWAL OF THE FISH SKELETOX 213 

lish without its ogfjs was found to be 17.78 mgm, or 1.17 per cent, while 
Ihe corresponding figures for the eggs were 2.7 and 0.29. 

Fish No. 16 hada weight of 1.07 gm including its eggs, which made up 
40 per cent of the total weight. TJver and heart were strongly degenera- 
ted, the liver weighing only a few milligrams. The duration of the experi- 
ment was 16 days, the activity of the eggs amounting to 12.8 per cent 
of the total activity. In the above case (No. 16 B), the corresponding 
ratio was 18.3. Thus, after the lapse of 16 days, only about 2/^ of the egg 
P was found to be incorporated in the eggs in the course of the experi- 
ment, while in experiment No. 16 B., after the lapse of 23 days, almosi 
the whole P content of the eggs was deposited (through growth or 
renewal) in the course of the experiment. 

In experiment No. 16 A., the percentage of P taken up in the course 
of the experiment from the water was found to be 1/3.9 • 10'* of the amount 
present, corresponding to 2.2-10"'^ y. 

In experiment No. 16 B, 1/2.2-10* of the water P was taken up by 
lhe fish amounting to 4.4 -10"^ y. 



DISCUSSION 

The average value for the degree of renewal of the fish skeleton in the 
course of 22 days was found to be 2.5 — 5 per cent, the lower value being 
the more probable. This means that, while 2.5 — 5 per cent of the skeleton 
were renewed once or several times — a part of this percentage was 
presumably renewed frequently — at least 95 per cent of the skeleton 
remained entirely unchanged. When arriving at this conclusion, we 
assumed that no additional growth of the skeleton took place in the 
course of the experiment. As such additional growth would take place 
from a labelled plasma, all newly formed skeleton might be expected 
to be labelled, and what we interpreted as a renewal of the skeleton 
might in such a case be due to additional bone formation in the course 
of the experiment. We could not find any evidence for a growth of the 
skeleton or a growth of the fish taking place in the course of the experi- 
ment. It is very difficult, however, to exclude the possibility of an in- 
crease in the mineral constituents of the skeleton by a few percent. The 
above mentioned 95 per cent therefore have to be considered a lower 
limit for the part of the skeleton remaining unchanged after three weeks. 
In the case of the fully grown rabbit, about 10 per cent of the skeleton 
were found to be renewed in the course of only 9 days (II eves Y et al. 
1940). The great difference in the renewal rate of the skeleton of the 
rabbit and that of the fish is presumably due to the great difference 
in the body temperature. The renewal of the skeleton is partly a "physi- 
cal" replacement process between the phosphate of the uppermost 



214 ADVENTURES IN RADIOISOTOPE RESEARCH 

molecular layer of the bone crystallites and the phosphate of the plasma» 
and. partly the effect of a "biological recrystallization". Crystallites 
or parts of crystallites go into solution and new crystallites are wholly 
or partly formed by crystallization from the plasma. In experiments 
of long duration, the interchange mostly takes place by biological recrys- 
tallization. Now, such a process may be expected to be strongly influen- 
ced by the body temperature and to take place at a higher rate at 37° 
than at 16°. 

It is interesting to note that in experiments of only a few hours' 
duration, increase of temperature was found to promote the radio - 
phosphorus uptake by the bones. The tibia of the frog (Hevesy et al. 
1940) was fo«nd to take up nearly II/2 times as much radio-phosphorus 
at 22° as at 0°. 

We found that a fish weighing about 1 gm took up, in the course of 16 
days, 1/4- 10* part the phosphorus added to the water i. e. 2- 10~^ mgm. 
As the water volume was 3000 ml., the amount of P taken up by the 
fish from water is equivalent to the amount of P present in 1/13 ml water. 
The amount of water taken up by the fish in the course of 16 days is 
presumably much larger than 1/13 of its body weight (cf. p. 206) and, 
thus, only a minor part of the P content of the water swallowed is absor- 
bed. 

Summary 

Sticklebacks (Gasterosteus aculeatus) were kept for periods of up to one month 
in 3 litres of sea-water containing labelled phosphate. A fish weighing 1 gm was 

found to take up in the course of 16 days part of the phosphorus present in 

the water, corresponding to 2 • 10"^ mgm P. 

By comparing the specific activity of the skeleton P with that of the free livei- 
P, figures for the degree of the renewal of the skeleton were obtained. At least 
95 per cent of the skeleton were found to remain unchanged during the experiment. 
The rate of renewal of the fish skeleton is thus much lower than that of the maiuma- 
lian skeleton. 

References 

G. Hevesy and L. Hahn (1938) Det Kgl. Danske Vid. Selsk. Biol. Medd. 14. 1. 
G. Hevesy, H. Levi and O. Rebbe (1940) Biochem. J. 34, 532. 
G. Hevesy and O. Rebbe (1940) Acta Physiol. Scand. 1, 171. 
K. Kjerulf-Jensen and E. Lundsgaard (1943) Ibid. 7, 209. 
A. Krogh (1939) O.smotic Regulatio?i in Aquatic Animals. Cambridge. 
R. S. Mani.y, H. C. Hodge and M. Le Fevre Manly (1940) J. Biol, f'hcni. 
134, 293. 

R. Robison The Significance of Phosphorus Esters in Metabolism. New York. 
I. Roche and M. Mourgue (1939) Bull. Soc. Chim. Biol. Paris 21, 143. 
F. Roth (1920) Anatom. Anz. 52, 513. 
H. W. Smith (1930) Awer. J. Phij-nol. 93, 480 (1931): Ibid. 8, 269. 



215 



Comment on papers 18—23 

The first application of an artificial radioactive isotope as a tracer in life sciences 
aimed to determine if and to what extent the mineral constituents of the skeleton 
of the fully grow-n rat are renewed. ^-P was used in this investigation (paper 18), 
which demonstrated the renewal of an appreciable part of the mineral constituents 
of the skeleton of the rat. The replaceable fraction of the bone phosphate was 
found to constitute about 30 per cent, a result which was confirmed by later inves- 
tigations. This study was carried out and its result published simultaneously- 
(1935) with ScHOENHEiMER and Rittenberg's first classical investigation on th(^ 
dynamic nature of fat deposits, followed by numerous others of a similai- type. 
The result that the formation of the bone is a dynamic process, involving conti- 
nuous loss and replacement was for many an unexpected and puzzling one. This 
is shown by a remark of the Editor of Nature. In those days, each issue of Nature 
contained a short survey of the contents of the "Letters to the Editor". In this 
survey it is stated: ". . . . The authors (Chiewitz and Hevesy) further believe (!) 
that the formation of the bone is a dynamic process involving continuous loss 
and replacement." A larger fraction of the epiphysial apatite molecules than 
of the diaphysial ones was found to be renewed, which is partly due to the fact 
that mineral molecules of the epiphysial tissue come more easily in contact with 
plasma and lymph containing the active phosphate than those of the more per- 
fectly mineralized diaphysial tissue. The first animals investigated included also 
rachitic rats (paper 19). At first the puzzling result that the fiaction of bone 
phosphate renewed in the rachitic rat was found to be larger than in the healthy 
animal, was at least partly due to the less well mineralized and thus more epiphysial 
nature of the rachitic skeleton. The uptake of minute amounts of ^^P by the enamel 
was found to take place even when preventing contact between enamel and sali\'a 
(paper 20). 

Since the specific activity of plasma phosphate diminishes with time, the 
sensitivity of the radioactive indicator correspondingly decreases (the same acti- 
vity indicates a larger amount of phosphorus). To arrive at an exact figure for the 
renewed fraction of the apatite phosphate, we must know the specific activity 
of the plasma inorganic phosphate throughout the experiment. An alternative 
method is to keep the plasma inorganic phosphate activity at a constant level 

apatite ^^p spec, activity 

throughout the experiment. The ratio — — X 100 determined 

plasma inorganic ^^P 

at the end of the experiment indicates then the percentage apatite renewal. By 
following the last mentioned procedure (paper 21), after the lapse of 50 days 
30 per cent of the femur epiphysis phosphorus, but only 7 per cent of the diaphysis 
phosphorus of the rabbit, was found to be renewed. A 44 per cent renewal of the 
scapula phosphate took place, and a full renewal of the apical and medial incisor 
phosphate occurred. At least two-thirds of the last mentioned renewals was due 
to incisor growth. The constancy of the serum-calcium and serum-phosphorus 
content of the plasma is maintained by homeostatic; mechanism, for which 
the skeleton is primarily responsible. The regulation is achieved mostly by a 
slight change in the amount of dissolved and newly formed bone apatite. 

While in the above mentioned investigations the dynamic nature of a substan- 
tial part of the skeleton apatite was demonstrated and the fraction of the latter 



21 6 ADVEXTURES IX RADIOISOTOPE RESEARCH 

which participates in a renewal process determined, information on more detailofl 
processes taking place in the skeleton were available only after the introdviction 
of the autographic methods of investigation in this type of study by Leblond 
ei al. (1951). 

The 32p applied in our early investigations was prepared under the action of 
neutrons emitted by radon-beryllium sources on 10 1. of CS^, the ^'^P being ex- 
tracted by strongly diluted acid. On his fiftieth birthday, Professor Niels Bohr 
was presented by his friends with 600 mgm of radium, which he most generously 
put at our disposal. It was the Union Haute Miniere which supplied the radium 
as sulphate mixed with 2 gm of beryllium. In view of the hygroscopicity of RaCl, 
the Union Haute Miniere was at that early date not willing to supply RaCl2- 
beryllium mixtures. After the availability of cyclotron-produced ^^p^ Professor 
Ernest Lawrence most kindly repeatedly mailed to us, starting in 1937, a few 
miUicuries of ^^p prepared by Dr. Martin Kamen. This was a very great help, 
as was 32p supplied later by Professor Bohr's and Professor Siegbahn's cyclotron. 

In paper 19, published in 1937, the first clinical investigation is describee] 
in which a radioactive isotopic indicator was applied, the determination to what 
extent the phosphorus of the faeces is of endogenous origin and the investigation 
of the incorporation of ^-P into the placenta. Paper 20, published in the same 
year, contains data on the incorporation of ^^P into humarr teeth, both into roots 
and crowns. Deuterium, thus a stable isotopic indicator was formerly used (paper 
54) in the determination of the water content of the human body. In 1937 Hamil- 
ton published a study on the rate of absorption of sodium by fasting human 
subjects following the oral administration of labelled sodium. Borsook et al- 
described in the same year the excretion by human subjects of administered ^^S. 



References 

C. P. Leblond, G. W. Wilkinson, L. F. Belanger and J. Robison (1951) 
Amer. J. Anat. 86, 289. 

H. Borsook, S. Keighley, D. N. Yost and E. McMillan (1931) Science 86, 
525. 

J. G. Hamilton (1937) Proc. Nat. Acad. Sci. U. S. 23, 521. 



Originally published in Z)a«6A(^ V idenskabernes Selskab Biol. Mcrhl. 22. no. 9(I95o) 

24. CONSERVATION OF SKELETAL CALCIUM ATOMS 

THROUGH LIFE 

(1 . Hevesy 

From the Institute for Research in Organic Chemistry, Stockholm 
Dedicated to Professor Niels Bohr on the occassion of Jiis 70lh birthday 

From the earliest beginning, Professor Niels Bohr has shown great 
interest in the application of radioactive indicators to the study of 
the conservation of skeletal atoms through life. This fact has induced 
the writer to contribute to this volume with a communication of the 
results obtained in an investigation on the conservation of skeletal 
calcium atoms in the adult mouse and on the fate of maternal calcium 
atoms through generations. 

The first application of an artificially radioactive isotope as a tracei- 
in 1934 was that of ^^p in a study of the problem whether and to what 
extent the mineral constituents of the skeleton of the adult organism 
are replaced during lifetime^^' ^' '^' ''\ By using this radioactive indicator 
it was possible to demonstrate the dynamic nature of the building u]) 
of bone tissue. It was found that an initial rapid location of the circu- 
lating labelled phosphate in the mineral constituents of the skeleton 
is followed by a slower second effect. The first effect was interpre- 
ted by us to be due to an interchange between the phosphate ions loca- 
ted in the surface layer of the bone apatite and in the plasma, the second 
one, however, to the fact that "the bone is destroyed at certain places 
and rebuilt under incorporation of labelled phosphate at others". Empha- 
sis was given to the analogy between these phenomena and those obser- 
ved when, in early experiments, naturally radioactive isotopes were 
applied as tracers. Paneth*^*\ when shaking solid sulfate with a solution 
containing labelled lead ions, observed an interchange of lead ions 
only between the uppermost molecular layer of the solid salt and the 
dissolved ions. In studies in which the interchange between the atoms 
of lead metal and the labelled lead ions of a solution, or vice versa, 
was investigated, the present author and others^'^' ^- '^ found that msbXiy 
hundreds Of atomic layers of the lead foil were converted into ions, and 
a corresponding number of ions into atoms, making out the lead foil. 
Thus, a renewal of the constituents of a metal foil, involving dissolution 
and reprecipitation due to "local currents", was found to be a much 
deeper going process than that occurring between solid lead salt and the 



•> ] 8 ADVENTURES IN RADIOISOTOPE RESEARCH 

lead ions of the sorrounding solution. In the early investigations mentio- 
ned above, it was pointed out that the rapid uptake of ^^p during the 
early phase of the experiment recalls the behaviour of a lead salt placed 
in the solution containing labelled lead ions, the recrystallization of the 
mineral constituents of the skeleton reminds of the behaviour of a lead 
foil immerged into a solution containing labelled lead ions, however, 
with the difference that, in the latter case, enzymic actions are involved. 
Or, as it was expressed later*'**, "A restricted extent of renewal of the 
skeleton is due to the fact that, while the P atoms of the uppermost 
molecular layer of the bone apatite crystals can promptly interchange 
with the free P atoms of the plasma (actually not the P atoms, but the 
phosphate ions interchange), a renewal of the main part of the apatite 
P can take place only when the crystal is dissolved and when new crys- 
tals are formed from the plasma; from labelled plasma, labelled crystals 
are formed". Subsequent experiments confirmed the correctness of these 
early conclusions, showing that both a surface exchange between plasma 
phosphate and bone phosphate, and a recrystallization, thus a dissolution 
of some of the apatite crystals and the formation of new ones, take 
place in the skeleton. Different workers, however, arrived at divergent 
results about the share of both processes in the interaction of plasma 
and bone constituents. 

The introduction of autoradiographic methods into the study of bone 
formation by Leblond et al.*^^\ was a very important advance, 
since it became possible to visualize the rapid formation and destruction 
of some parts of the calcified tissue. Numerous autoradiographic investi- 
gations such as those by Leblond et al. applying ^^p^ those by 
(JOMAR etal.^'''^ using 45Ca, ^aSr, and 32p, by Skipper etal.^"^ with i^C, by 
Kidman et al.*^^"^ with ^^Sr, by Engfeldt et al.^^''* with ^^p^ by Amprino 
and Engstrom*^^*^ with 45Ca, and by Bauer^^^^ with 22 Na, clearly demon- 
strate that a great part of the bone salt crystals are more or less unchan- 
ged until they are reached by the process of resorption. 

Leblond's autoradiographs clearly indicate that the circulating 
phosphate enters the skeleton either by exchange or by precipitation in 
definite areas with the formation of new bone. While, in the autoradio- 
graphs, the exchangeable phosphate is depicted as diffuse reactions 
disappearing rapidly with time, the precipitated or stable phosphate 
appears as localized persistent reaction. 

In contradistinction to all workers in this field, Engfeldt, Engstrom 
and Zetterstrom^^ arrived at the result that even the initial uptake 
of 32p by the bone is due exclusively to some kind of recrystallization. 
This conclusion is based on their observation that the autoradiographic 
patterns of cross sections from long bones show an uneven distribution 
of radioactive phosphate. They found the fastest uptake of labelled 
phosphate to take place in Haversian systems with a low content of mine- 



CONSERVATION OF SKELETAL CALCIUM ATOMS THROIOH LIFE 219 

ral salts. Since the major part of the tracer is found in limited areas, 
the initial rapid uptake of labelled phosphate — according to 1heir 
view — cannot be due 1o ion exchange on the crystal surface of the bone 
minerals, such an exchange being prevented by the organic constilu- 
cnts of the bone. 

While there can hardly be any doubt that the main part of the renewal 
of the bone apatite of skeleton is due to a recrystallization process, 
to a degradation and new formation of the mineral constituents of the 
skeleton, objections may be raised against the view that the initial 
uptake of ^^p is due exclusively to some kind of recrystallization. 

Uneven distribution of radioactive phosphate as shown in auto- 
radiographic patterns of cross sections from long bones cannot be inter- 
])reted as an absence of surface interchange. According to Paneth*^^- ^"^ 
th(> whole uppermost molecular layer of crystalline salt powders inter- 
changes with the ions of a surrounding solution, while properly crystal- 
lized surfaces like those of natural crystals fail to do so. He states that 
his investigations suggest that the radioactive method of determining 
surfaces, based on the assumption that the whole uppermost layer 
molecular interchanges, should be employed in those cases only for 
which it is established that the fundamental supposition of kinetic 
exchange of the entire surface is valid. If we assume the bone apatite, 
or part of it, as occurring in vivo, to be a properly crystallized substance, 
Ave arrive at another explanation than that of Engfeldt, Engstrom 
and Zetterstrom, according to which the organic constituents of 
fresh bone are responsible for preventing surface exchange. This alter- 
native explanation is that the bone apatite, or large parts of it, behaves 
like a properly crystallized substance in Panetli's experiments and not 
like a crystal powder. 

The exchange of ions on a crystal surface is thus far from being absent 
and, though restricted to a fraction of that surface, is responsible for 
an appreciable part of the early uptake of labelled ions by the mineral 
constituents of the skeleton. As shown by Armstrong and assoc.^^"\ 
in the course of the first ten minutes, 2% of the skeletal calcium of the 
dog are replaced by labelled calcium of the plasma; this is Yio oi^ly o^ the 
amount which, according to Falkenheim's^^*^' ^^''^ calculations, would 
be necessary to replace the whole uppermost molecular layer of the bone 
apatite, or i/g of the amount estimated by Hendricks and Hill^^'". 
A large part of these 2% — or even 2% — could be due to a surface inter- 
change in spite of the uneven autoradiographic patterns of cross sections 
of long bones observed by Engfeldt, Engstrom, and Zetterstrom. 
Tn view of the high specific activity of the plasma calcium in an early 
stage of the experiment, to a 2% interchange corresponds a verj^ much 
higher percentage decrease in the ^^Ca content of the plasma in the 
course of the first two minutes, more than 50% of the injected radio- 



22f> ADVENTURES IN RADIOISOTOPE RESEARCH 

calcium leaving the circulation. From Ca*^ injected into the circulation 
of growing hogs, Comar and assoc/^'^^ found only 2% to be present 
after the lapse of an hour. 

In the experiments of Engfeldt and assoc, three hours was the 
shortest time after which the ^^P injected rats were sacrificed. The early 
phase of the experiment, in which a very rapid interchange of the mineral 
constituents of the bone takes place, is much shorter than three hours. 
When investigating the uptake of *^Sr by the skeleton of outgrown 
rabbits during the first 30 seconds, 11.7% were found to be taken up*^-"*, 
while during the first six hours— thus a 720 times longer period — only 
about twice as large an uptake was observed. Armstrong and assoc. ^^'* 
found during the first 20 minutes an interchanges of 4% of the skeletal 
calcium with plasma calcium, this amount increasing less than three 
times during the following 160 minutes. 

A change in the concentration of the bone apatite constituting ions, 
and still more a variation in the concentration of enzymes involved 
in recristallization of the plasma and the lymph, is bound to influence 
the rate of recrystallization of the skeleton. Repeated administration 
of bone phosphate extract by intravenous injection was found to lead 
to a decrease of the mineral constituents of the bone tissue*^^^\ which are 
replenished after removal of the excessive phosphatase. Hastings^"', 
when replacing the plasma of a dog by plasma of low calcium content, 
found that the mobilization of bone calcium increased the calcium level 
of the plasma almost momentarily to a normal level. Parathyroid hor- 
mon is known to exert a direct action on bone*^'^^'''\ In this connection, 
also Carls ON 's*^^^^ investigation should be mentioned; he found vitamin 
D deficient rats to be unable to utilize their bone stores for maintaining 
a normal serum calcium. However, in view of the very great difference 
in the distribution of the mineral constituents in the bone tissue and the 
corresponding tendency to remove these differences, the biological 
recrystallization of the skeleton, as rightly emphasized by Engfeldt 
and assoc .^^^\ is not due exclusively to these processes^^'*\ 

King raised the idea that, though conventionally, the bony frame- 
work of the body is regarded as a means of making locomotion possible, 
it may be that this is no more than a secondary development, the pri- 
mary function of bone in the body being to act as a reservoir for the 
maintenance of a constant blood calcium level. 

At a very early date ■^^' ^^' ''^ it has already been observed that the 
diaphysial phosphate is replaced by administered labelled phosphate 
at an appreciably lower rate than epiphysial phosphates, and similar 
observations were made in the investigation of the incorporation of 
*^0a into the skeleton^^^' ^^\ Since the transition between diaphysial and 
epiphysial bone tissue is almost continuous, the specific activity of 
bone phosphorus or bone calcium varies considerably through the whole 



i) 



CONSERVATION OF SKELETAL CALCIUM ATOMS THKOlfiH LIFE 221 

bone tissue. This great heterogeneity of the specific activity of the bone 
apatite phosphorus could be demonstrated by Zetterstrom and Ljung- 
(iREN*^^^^' by isohiting bone fractions of different solul)iHty and measuring 
Hieir specific^ activity. The most soluble bone phosphorus was found 
lo show the highest specific activity, thus the most rapid rate of renewal. 
X-ray absorption and diffraction studies by Amprino and Engstrom*^' 
revealed also thai the distril)ution of mineral components in the bone 
1 issu(^ is far from being uniform. 



SIZE OF THE NON- RENEW ABLE PART OF THE SKELETON 

The extent of renewal of apatite phosphate of the skeleton can be 
calculated from the mean value of the specific activity of the plasma 
l)hosphate during the experiment and the value of the specific activity 
of the apatite phosphate at the end of the experiment. During the ear]3^ 
part of the experiment, the sensitivity of the radioactive indicator is 
comparatively low, thus a strong decline in the plasma activity corres- 
ponds to a comparatively low interchange figure. In the later part of 
the experiment, the same activity which indicated at the start the pre- 
sence of 1 mgm of phosphorus in the plasma, for example, indicates 
100 mgm, thus the sensitivity of the radioactive indicator is strongly 
increased. Now, a further interchange will be indicated by a very small 
further loss of activity. Furthermore, following the interchange of 
l)lasma and bone phosphate for a longer time interval, increase and 
decrease in the specific activity of the plasma phosphate may alternate 
due to a variation in the phosphate intake or other reasons. Thus, it 
encounters difficulties, by comparing the mean specific activity 
of the plasma phosphate during the experiment and the specific activity 
of the apatite phosphate at the end of the experiment, to find a reliable 
value for the extent of the renewable part of the mineral phosphate 
of the skeleton, and similar considerations apply to the determinatior 
of the renewable part of bone calcium in contrast to that of the bone 
sodium. Sodium, being mainly an extracellular element, is distributed 
between plasma and extracellular fluid within a few minutes, a distri- 
bution wdiich results in a decrease in the specific activity of plasma 
sodium to about i/g of its original value, followed by a slow decrease 
with time only. Thus, as discussed on p. 16, the extent of the renewable 
part of the mineral bone sodium could be calculated from specific acti- 
\'\\y data. We can, however, determine the extent of renewal of bone 
phosphate from specific activity data when keeping the specific activity 
of the plasma phosphate at a constant level during the experiment. 
This result was oblainerl bv the author and his associatrs^i by daily 



222 ADVENTURES IN RADIOISOTOPE RESEARCH 

injecting the rabbit repeatedly with labelled phosphate. After the laps 
of 50 days, the phosphorus of the femur epiphysis found to have a spe- 
cific activity of 30% of that of the plasma inorganic P, thus indicating 
that 30%, and only 30%, of the epiphysial bone apatite had been rene- 
wed, a much lower renewal figure (7%) being obtained for the diaph3^sial 
phosphorus. 

This method has the disadvantage of being cumbersome. Furthermore, 
the results may be influenced by the time that passes between the last 
injection of the rabbit and the killing of the animal. Therefore, when 
determining the renewable fraction of the skeleton calcium of the mouse, 
we have chosen another procedure. Mice were bred whose skeleton was 
labelled throughout with ^^Ca and the loss of the activity in the skeleton 
was followed with increasing age of the animals. Such mice can be 
obtained by administering to the mother food containing labelled cal- 
cium already weeks before gestation and continuing to feed the lactating 
mother and the growing offspring with food containing labelled calcium. 
We assume every one of the offspring to have the same '*^Ca content. 
If we stop administering labelled food after these offspring are out- 
grown, they start to interchange their labelled bone calcium with the 
unlabelled calcium from the food with the result that the *^Ca content 
of the skeleton decreases and, when the offspring is killed after two 
months, its *^Ca content is lower than that of another offspring killed 
after one month. By killing members of a litter at different dates, we 
can follow the processes in the skeleton for years, viz. through the 
lifetime of the animal. 



Table 1. — Weight and Activity 
OF New-born Mice 



No. 


w 

! 


eight ill 


Kin 


Relative 
iictivity 


1 


1 


1.8 




100 


2 




1.3 




98.5 


3 




1.4 




93.5 


4 


i 
1 


1.2 




99.5 



The •*^Ca content of every member of a litter is not strictly the same, 
and this applies also to the growth rate. The evidencee that a part of the 
curve depicted in Fig. 1 (p. 224) is discontinuous may presumably be due to 
a difference in the uptake of maternal '^^Ca by the offspring of the same 
litter. The variation in the radioactivity of different members of a litter, 
however, is restricted and does not suffice to frustrate the applicability 
of the method described (cf. Table 1). 



COXSEHVATIOX OF .SKELETAL CALCHM ATOMS TllKOUCll LITE 22:i 

EXPERIMENTAL 

In view of the dill icult i(>s in r(>pluc'ing all food ciUciuni hy ialjollod oalciuin. 
wo added the labelled caleium as CaCl^ (150 mgm. per liter) to tlu^ drinking wa1(M-. 
on the assumption that the quantity of water drunk by tlie nions(>, kept at (con- 
stant teni])eratuie, is about proportional to the intake of food which consisted 
of standard cakes. We started to administer two to ten weeks before parturition 
to 20—30 gm mice the labelled CaClg nnd continued administration of such 
drinlving water till weaning. Then, the growing mice were given labelled drinking 
water until they were outgrown. From that date (when the mice were aljoul 
100 days old), administration of *^Ca was discontinued. The offspring were killed 
at different times, and the radioactivity of the ash of their skeletons was compared- 
20 mg. of bone ash were placed under the Geiger counter, and the total activity 
of the skeleton was calculated fiom the measured activity and the total ash weight. 
In other experiments, the radioactivity of the total body ash samples was com- 
pared. 

The ratio of the activity of 20 mgm of Ijone asli of outgiown and of newborn 
mice is not a correct measure of their relative *^Ca content. The calcium content 
of the ash of the newborn being appreciably lower than that of the adult, the 
backseat tering of the j^-rays emitted by the ^^Ca of the first mentioned samples 
will be lower, furthermore the consistency of the samples and, thus, the distance 
of the sample from "the counter window may slightly differ. By measuring once 
the activity of a 20 mgm sample of the bone ash of newborn mice, and then that 
of a small known aliquot of this sample brought up to 20 mgm through addition 
of inactive bone ash of an adult mouse, wt arrive at the result that the activity 
measured of the ash of the newborn mouse has to be multiplied by 1.05 in order 
to make it comparable with the activity of the bone ash of adult mice. 

In other experiments, new-boin mice were shifted from their active mothers 
to inactive mothers shortly after birth; determinations were made of the percent- 
age of maternal labelled calcium taken up by the offspring after birth and the 
rate of loss of these calcium atoms during growth and later. 

The ^^Ca activity of the mice remained below 0.05 ^C per gm and, in most 
cases, it was very appreciably less. Simmons and assoc.^^-^ observed the effects 
of radiation produeed in mice during 108 weeks. When a dose of 0.034 juV per gm wa , 
administeied, they could not find anaemia; when the dose was laised to 0.068 juC 

Table 2. — Composition of Cakes Fed to orR Mice ("Gard-kred")* 

100 gm cakes (.■outain 



Water 

Ash 

Proteins (.l.T >( N) 
Carbohydrates . . . 

Ca 

P 

Fe 



Combustion value (calculated according to Rubner) .... 400 cal. 



7.0 


t;m 


2.9 


?3 


6.3 


.. 


67.6 


J3 


ISS mgm 


424 


? J 


24 





* In Sweden, mice and rats are fed almost exclusively on these cakes, the exact composition of wliicli 
was hitherto unknown. The author is much indebted to Professor E. BRrNU's and Mrs. EsTHKR SIHLBOM 
wlio most kindly made the analysis of these cakes at Statens Institut for Kolkhiilsan. 



224 



ADVENTIKES I.V RADIOSIOTOPE RESEARCH 



moderate changes in heteiophylous values could be detected. In our experiments, 
no effect on growth or fertility due to the presence of ^^Qa could be observed. 
Our main litter size was 5.7. As shown by Russell(^^\ the litter size of mice at 
term is reduced as a result of irradiation during preimplantation stages with 
100 r or more, and when exposed shortly after implantation, by a minimum dose 
of 200 r. 

The composition of standard cakes fed to our mice is seen from Table 2. 



RESULTS 

The results of experiments in which mice born from mothers kept 
on a *^Ca diet for weeks prior to and after parturition, and continuously 
kept on a ^^C'a diet till they reached an age of about 100 days, thus 
were fullygrown, are shown in Table 3. 

The mean conservation of *^Ca by the uniformly labelled skeleton 
of the mice in the course of 390 days, representing a mean value of the 
duration of the experiments, works out to be 64.7 ± 7.34 per cent, the 
standard error of the mean being 2.78. If we disregard the last experi- 
ment in which the mice were kept on a high calcium diet, the mean 
value is 67.2 ^ 7.86 per cent, the standard error of the mean being 
3.23. Thus, -/g of the calcium atoms present in the skeleton of the out- 



o 
U 




too 



200 



300 
days 



400 



SCO 



600 



Fig. 1. Loss of *^Ca, obtained from labelled mother at biith, during 

lifetime of the mice. Each point indicates the ^^Ca content of 

another member of the litter killed at the stated time 



COXSEKVATIOA' OF SKELETAL CALCHM ATOMS THKOl (iH LIFE 



225 



Table 3. — Loss ok ^^Ca «y the Uniformly Labelled 
Skeleton of Mice with Time Indicated by Measurements ok 
THE Radioactivity of the Skeleton of Different Mkmbkks 
OF a Litter Killed at Various Times. The Mice wkre Born 
FROM Active Mothers and were Administered ^^Ca until the 
First Member of the Litter was Killed 



No. of Utter 



II 



III 



IV 



V 



VI 



VII"" 



Age in days '^(ja content 



111 


100 


329 


66.7 


519 


57.0 


108 


100 


327 


00.7 


517 


78.8 


108 


10(t 


326 


88 


501 


69.4 


115 


100 


220 


79.9 


393 


64.4 



106 
106 
231 
325 

56 
129 
266 

99 
214 
308 
392 
503 



100 
66 
63.8 

100 
81.4 
60.7 

100 
77.7 
55.5 
55.9 
50.1 



* Cheese and egg shells were added ad lib. to the standard "gard-bred" diet. 

grown mice are present after the lapse of more than a year and can tlius 
be considered to be unreplaceable during life. 

Figs. 1 and 2 and Table 4 show the results of some of our experimenis 
in which the litter, born from active mothers, was kept from birth on a 
^Ca-free diet. These experiments include the results obtained between 
the third and the 560th day after birth, thus almost the lifetime of the 
mouse. The percentage of ^^Ca lost between day 3 and day 560 works 
out to be 53 per cent and 44 per cent, respectively. The mean loss of 



15 Hevesv 



226 



ADVENTURES IN EADIOISOTOPE RESEARCH 



^^Ca observed in experiments lasting 100 to 180 days amounts to 43 
per cent (Table 4). The loss of ^^Ca during the first three days of life 
is less than 10 per cent; thus half of the maternal calcium atoms are 
preserved during life. 



100 -rt 



80- 



c 

V 

c 
o 

u 

o 
u 



60- 



40- 



20 



~1 — 
100 



200 



300 



400 



500 



600 



doys 



Fig. 2. Loss of *^Ca, obtained from labelled mother at birth, during 
lifetime of the mice. Each point indicates the ^^Ca content of another member 

of the litter killed at the stated time 



The calcium content of our newly born mice, weighing 1.23 — 1.37 
gm. varied between 0.28 and 0.35 per cent of the body weight, not 
much differing from the calcium content of the new-born rat (4.7 gm) 
for which data varying between 0.27 and 0.35 per cent are reported*^^^\ 
The calcium content of 1 gm fresh weight of newly born mouse amounts 
to 0.3 times that of 1 gm of the adult animal, which is 1.05 per cent. 
If all the maternal calcium atoms had the same chances to supply cal- 
cium to the offspring, and all were labelled, we would find 1 gm of 
newly born mouse to be 0.3 times as active as 1 gm of the mother. 
We find the ^^Ca content of 1 gm of new-born mouse to amount to 1.7 
times that of 1 gm of the adult mouse. The ^^Ca taken in by the mother 
has thus only an opportunity of interchanging in the average with 
about 1/5 to i/e of ^^^^ body calcium before being utilized in the building 
up of the embryo. 



rOXSERVATIOX OF SKELETAL CALril M ATOMS niUOrcir LIFE 



227 



Table 4. — Rktkxtiox of Materxal Cai^cium Atoms by 

THE OkKSPRIXGS 



Xo. of litU'i- 



Age in days 



iu j?m 



Per cent oi 

motliers' activity 

present in the 

offspring 



X'uniber of 
maternal atoms 
present 



II 



III 



IV 



V 



3 


2.95 


31 


13.9 


39 


16.8 


43 


18. G ! 


103 


24.3 


1 


2.20 : 


129 


41.9 


181 


31 


1 


1.80 


131 


38.1 


1 


1.45 i 


128 


39.6 ' 


181 


40.0 


3 


3.1 


27 


11.5 


35 


21.0 


42 

1 


24.2 i 



S.25 

t).59 

5.50 

6.0 

4.25 



KtO 
80 
67 
72 
52 



6.95 


100 


4.95 


71 


3.96* 


57 


0.3 


100 


7.4 


72 



9.91 
7.25 
5.69 

8.55 
6.98 
6.67 
6.27 



100 
73 
57 

100 

82 
78 
73 



DISCUSSION 

a) Conservation of the calcium atoms of the outgrown skeleton through 
life 

The fact that a very appreciable part of the skeletal calcium is preser- 
ved in the outgrown animal throughout its lifetime results from experi- 
ments carried out by Singer, Armstrong and Premer^^^\ by (J arlson^^^' ^'^^ 
and by Bauer^'^'^^ Similar results were obtained in investigations on 
the renewal of the mineral constituents of the skeleton, performed by 
the present author and his assoc. who used ^ap as an indicator^''^^ 

From specific activity data of the plasma and the skeleton of the 
outgrown rat, the percentage of renewable skeletal sodium was calcu- 
lated by Bauer^^^*^ to amount to 30—40 per cent of the sodium present 
(disregarding the extracellular sodium); a similar figure — 45 per cent — 
is reported by Edelman^^*^^ and by Baden and Moore^-'"'\ Since sodium 
is mainly an extracellular element, the specific activity of plasma sodium 
decreases only slowly with time, not so the specific activity of calcium. 
The calculation of the percentage of renewable skeletal calcium from 



15* 



228 



ADVEXTUKES IX llADIOISOTOPE RESEARCH 



specific activity data is therefore encumbered with difficulties 
(cf. p. 221). From data collected during five days, Bauer*'^* estimates, 
however, that less skeletal calcium than skeletal excess sodium is exchange- 
able in the rat, thus less than 30 — 40 per cent. As it was shown above 
(p. 217), the mobilization of some further skeleton calcium is still going 
on in the mouse after the lapse of more than 100 days and the non- 
exchangeable part of the skeleton amounts to 67 per cent. 

It is interesting to note that, w^hen injecting ^^fja at the start of the 
experiment interperitoneally to outgrown rats whose skeletal calcium 
content was increased appreciably during the experiment, Singer and 
Armstrong^*^^ found a *^C'a retention of 42—45 per cent in the skeleton 
after the lapse of 52 days and the release of only small amounts of 
radiocalcium after that date. Buchanan'^^^\ who exposed mice to air 
containing ^K!0^, found that 30 per cent of the bone carbonate are 
replaced within 12 days, while 45 per cent only are renewed in the 
course of three months. 

b) Conservation of maternal calcium atoms by the offspring through life 

Our results demonstrate the very pronounced ability of the skeleton 
1o conserve maternal atoms. 

In the first mentioned experiments, one third of the ^^C'a content of the 
outgrown mouse was found to be replaceable by inactive food calcium. 
In the latter experiments with growing mice, released "^^Ca had a furthei- 
outlet, viz. utilization in the formation of additional skeleton, which 
takes place in the growing organism. 

Investigations were carried out earlier on the loss of ^^p through the 
lifetime of mice born from active mothers'"''. Some results of these 
investigations are shown in Table 5. 



Table 5. — Loss of ^^p through the Lifetime 

OF Mice Born from Active Mothers. Mother 

Injected With ^-P on Febri^ary 0. Gestation: 

February 18. Replacement of the Active by 

AN Inactive Mother: Febritary 22. 



Ko. of offspring 


Killed : 
date 


KeUitive 
activity 


Weight 
in gm 


1 

-> 


22/2 
3y3 
l«/3 
30/3 
13/4 

13;:") 


100 
82 
73 
4« 
41 
40 


3 

7 


3 

4 


15 

18 


.-, 


25 


(i . 


35 









* Incl. G" or 3 otTsprinsj 



COXSERVATIOX OF SKELETAL CALCIUM ATOMS TIIROUCiU LIFE 229 

The fact that a very appiecial)l(5 percentage of the maternal phosphalc 
is preserved ^though less than of the mal(M-nal calcium— is presumably- 
due to the lower share of the hone ])h()sph()rus in the total body phos- 
phorus than the part of bone calcium in body calcium. 17 per cent of 
th{^ phosphorus content of the mouse are present in Ihe soft tissues, 
but only 1 per cent of its calcium content is located there. The phosphorus 
and calcium atoms present in various components of the soft tissues — 
with the exception of desoxyribo nucleic acid phosphorus of some tis- 
sues—are poorly conserved and, consequently, maternal calcium may 
be expected to be better conserved than maternal phosphorus. 

From the fact that during the first 40 days of life — thus during a 
phase of intense skeleton formation— only less than a third of the mater- 
nal calcium atoms of the mouse is lost, we can conclude that the largest 
part of the calcium atoms leaving the circulation is utilized to skeleton 
formation and remains largely conserved in Ihe skeleton. 

Leblond and assoc.'"^ injected labelled phosphate into newborn 
rats and followed the ^^p uptake by the humerus and the lower jaw. 
Denoting the total ^^P taken up by the humerus in the course of the first 
hour by 100, the uptake after eight hours was found to be 150, after 
one day 117, and after three days 116. In spite of the rapid growth of 
the humerus, the ^^p present after the lapse of a day is thus conserved 
through the following days; similar results were obtained in investiga- 
tions on the ^^P uptake by the lower jaw. 

The incorporation of calcium atoms in the rapidly growing bone tissue 
can also be studied by following its uptake into the incisor of outgrown 
animals. Carlson^"'"'"'^' ^'"^^ performed extensive and highly instructive 
studies on the calcium metabolism of outgrown rats, among others 
with the result that the calcium atoms incorporated with the rapidly 
growing incisors are conserved to a very large extent in contrast to 
those incorporated with the outgrown skeleton. 

It is rather difficult to determine the calcium intake and excretion 
by the suckling mouse. Our adult mice (36 — 37 gm), however, were 
found daily to consume 4 i 0.6 gm of standard bread containing 
8.3 + 1- mgm. calcium; further 0.2 mgm calcium was contained in the 
4 ml. of daily consumed water. The calcium recovered daily in the faeces 
amounted to 8 mgm. A very appreciable part of the faeces calcium may 
be assumed to be of endogenous origin, thus having passed th(^ circulation 
before excretion. The share of endogenous phosphorus in the faeces 
phosphorus was calculated from the specific activity of faces P and 
urine (plasma) P*^^^''*^) these calculations lead to the result that 74 per 
cent of the phosphorus of the human food and 72 per cent of the rat 
food are absorbed into the circulation. About the same percentage of 
the food P can be expected to be taken up by the mouse. As to the utili- 
zation of calcium, data are available only for the uptake by luimans^^'\ 



230 ADVENTURES IN RADIOISOTOPE RESEARCH 

Here, the mean percentage uptake was found to be 56. From the above 
data it follows that, out of the daily uptake of 8 mgm calcium by our 
mice, at least 4 mgm. have passed the circulation, representing a mini- 
mum amount of 2 mgm in the course of 500 days. From our results it 
thus follows that these 2 mgm were prevented from interchanging with 
2/3 of the 370 mgm calcium present in the skeleton of a mouse weighing 
36 gm. The protected part of the skeleton calcium did not come into 
contact with the plasma or lymph and, correspondingly, an exchange 
between the unlabelled food calcium and labelled skeleton calcium could 
not take place; the same is true for the new-formation of the protected 
apatite crystals of this part of the skeleton under participation of food 
calcium. A possible rearrangement within the protected area would 
not manifest itself in our experiment. 

The inaccessibility of parts of the skeleton minerals manifests itself 
also by the observation that radium, which like calcium is a strongly 
bone-seeking element, can find a life-long abode in the skeleton. The 
fact that a large fraction of radium administered to human subjects 
remains for decades in the skeleton is due presumably to the incorpora- 
tion of the radium into parts of the skeleton which are covered by apatite 
layers and thus become inaccessible and, even if released, are incorpo- 
rated again with the apatite structure. Aub and associates'^"' report 
a case in which no decrease in the radium content of a woman was found 
to take place between 1934 and 1945. This woman had been administered 
radium in 1924. 



CONSERVATION OF ANCESTORAL ATOMS 

The radiocalcium atoms going over from the first generation of mice 
into the second (cf. p. 226) do not indicate the total amount of maternal 
calcium atoms passing from the mother to the offspring, since the mother 
is not uniformly labelled. Chemical data indicate a passage of about 
1 .3 per cent. Since the calcium of the second generation is uniformly 
labelled, the passage of the ancestoral calcium atoms from the second 
into the third generation is properly indicated by the radioactive tracer. 
Aliout one third of the *^C'a content of the second generation is lost 
prior to gestation, while about 0.5 per cent or less of the remainder 
passes into the third generation. From the calcium atoms present at 
birth in each generation, thus 1/300 P^''^ °^" ^^'"^^ §°^^ over to the following 
generation. As our mice contained 6 . lO'-i calcium atoms, the eleventh 
generation did no longer contain a single ancestoral calcium atom. 

It is of interest to compare the life cycle of the ancestoral calcium 
atoms of the mouse with that of easily accessible water molecules. 
Applying deutoriatecl or tritiated water as an indicator first the half 



CONSERVATIOX OF SKELETAL CALCIUM ATOMS THROUGH LIFE 231 

life of water molecules present in the rat was found to vary between 
3.6 and 2.5 days*^-'"''' '^^^ that of the mouse is expected to be somewhat 
shorter. Thus, in the course of 165 days, all 10^4 water molecules present 
at the start of the experiment in the mouse are replaced. About 4 per 
cent of the maternal wattn- molecules go over to the offsprings and, 
from these, the second and third generations of offsprings will take up 
a share which depends on the age of gestation; the fourth generation, 
however, will hardly contain any more ancestoral water molecules. 

When the rate of disappearance of labelled water was followed in the 
rat during a long period, which was made possible by using tritiated 
water as an indicator, it was observecP'^^ that, after the lapse of 30 days, 
the labelled water disappeared at an appreciably slower rate than with 
a half- life of 2.5 days. The controlling factor of the dissappearance of 
labelled water from the organism is now the release of firmly bound 
tissue tritium which again becomes a constituent of the water molecules. 
Due to this fact, it lasts 60 days until the number of labelled water 
molecules of this type, present in the mouse, decreases to a 10""^th of 
its initial value. 

If we disregard those water molecules whose hydrogen atoms were 
temporarily incorporated in tissue constituents and released appre- 
ciably later to become constituents of water molecules again, then all 
ancestoral water molecules are lost by the mouse during two generations. 

While the loss of ancestoral calcium is determined mainly by the loss 
at birth, many ancestoral water molecules are lost during the lifetime 
of a generation, none of them reaching the third generation of offsprings. 



Summary 

Since it was desirable to obtain uniform labelling of all calcium present in the 
.skeleton of the mouse, ^^CaClg was added to all water administered to mice for 
weeks before and after gestation. Such water was also given to new-born mice 
after weaning until adult age was reached. The members of the litter, having 
almost the same radiocalcium content, were then sacrificed at different dates 
within 560 days. 

From the labelled calcium atoms present in the skeleton of the outgrown 
mice, 67.2 ^b '^•9 P^r cent were found still to bo present in the skeleton of sister 
mice sacrificed after the lapse of 390 days. 

When administration of ^^Ca was interrupted after the birth of the litter, and 
its members reared by inactive mothers were sacrificed at different dates within 
560 days, a mouse killed shortly after birth contained 8 per cent of the maternal 
^^Ca atoms, another mouse killed after 510 days contained 4 per cent. Half of the 
calcium atoms present at birth is thus conserved during the lifetime of the mouse. 

From the figures obtained of the passage of labelled calcium from one geneia- 
tion to the next, it follows that the eleventh generation does not contain a single 
calcium atom present in the first generation of its ancestors. 



282 ADVEXTURES IX RADIOISOTOPE RESEARCH 

References 

1. O. Chievitz and G. Hevesy, Nature 136, 7o4 (1935). 

2. O. Chievitz and G. Hevesy Dan. Biol. Medd. 13, No. 9 (1937). 

3. M. Manly and W. F. Bale, J. Biol. Chem. 129, 125 (1939). 

3a. R. S. Manly, H. C Hodge, end M. Manly, J. Biol. Chem. 134, 293 (1940). 

4. F. Paneth, Z. Elekirochem. 28, 113 (1922). 

5. G. Hevesy, Phys. Z. 16, 59 (1915). 

6. G. Hevesy and M. Biltz, Z. phys. Chem. B 3, 270 (1929). 

7. O. Ebbacher, Z. phys. Chem. A 166, 23 (1933). 

S. G. Hevesy, Los Prix Nobel, Stockholm 1940—1944, p. 95. 
9. C. P. Leblond, G. W. W. Wilkinson, L. F. Belanger and J. Robison, 
Amer. J. Anat. 86, 289 (1950). 

10. C. L. Comar, W. E. Lotz and G. A. Boyd, Amer. J .Anat. 90, 113 (1951). 

11. H. E. Skipper, C. Nolan and L. Simpson, J. Biol. Cem. 189, 159 (1951). 
J 2. B. Kidman, B. Rayner, M. L. Tutt and J. M. Vaugham, J. Pathol, and 

Bact. 64, 453 (1952). 

13. B. Engfeldt, a. Engstrom and R. Zetterstrom, Biochim. et Biophys. 

Acta 8, 375 (1952). 

14. R. Amprino and A. Engstrom, Acta Anat. 15, 1 (1952). 

15. G. C. H. Bauer, Acta Orthop. Scand. 23, 169 (1954). 

I(). F. Paneth, Radio-Elements as Indicators p. 55. MeGraw-Hill, New Yoik 

(1928) 
17. W. D. Armstrong, J. A. Johnson, L. Singer, R. I. Lienke and M. L. 

Premer, Amer. J. Physiol. 171, 041 (1952). 
IS. M. Falkenheim, W. F. Neuman and H. H. Carpenter, J. Biol. Chem. 

169, 713 (1947). 
18a. M. Falkenheim, F. E. Underwood and H. C. Hodge, J. Biol. Chem. 

188, 805 (1951). 

19. S. B. Hendricks and W. D. Hill, Metabolic Interrelations p. 141. Josuah 

Macy Jr. Foundation (1951). 

20. M. Tutt, B. Kidman, Brayner and J. Vaughan, Brit. J. Exp. Pathol. 

33, 207 (1952). 

21. H. G. Hodge, E. Gavett and J. Thomas, J. Biol. Chem. 163, 1 (1946). 

22. B. Hastings, Metabolic Interrelations p. 40. Josuah Maey Jr. Foundation 

(1951) 
22a. M. A. Logan and P. O'Connor, J. Biol. Chem. 127, 711 (1939). 

23. A. Carlson, Acta Physiol. Scand. 31, 30 (1954). 

24. G. Hevesy, Isotopic Indicators p. 421. Interscienee Publishers, New York 

(1948). 

25. L. A. Hahn, G. Ch. Hevesy and E. C. Lundsgaard, Biochem. J . 31, 1705 
(1937). 

26. M. J. L. DoLS, B. C. P. Jansen, G. J. Sizoo and G. J. van der Ma.'V.s, Koninkl. 
Nederl. Akad. Vetenschap. Proc 42, No. 6, 1 (1939). 

27. H. E. Harrison and H. O. Harrison, J. Biol. Chem. 185, 857 (1950). 

28. A. Carlson, Acta Pharmacol. 7, Suppl. 1 (1951). 

29. W. Minder and T. Gordonoff, Experientia 8, 71 (1952). 

30. O. Zetterstrom and M. Ljunggren, Biochim. et Biophys. Acta 8, 283 (1952). 

31. G. Hevesy, H. Levi and O. H. Rebbe, Biochem. J. 34, 532 (1940). 

32. E. L. Simmons, L. O. Jacobson, E. K. Marks and E. Lorenz, Badiology 
52, 371 (1949). 

33. L. B. Russell and W. L. Russell, J. Cell. Comp. Physiol. 43, Suppl- 1, 
103 (1954). 



("ONSBRVATIO.N OF SKELETAL CALCIUM ATOMS THKOllill IJl'E ^'.VA 

:\4. I.. Six(iEH, \V. I). Akmstrong and M. L. Prkmeb, Prcc. Soc. Exp. Biol. Mai. 
80, G43 (1952). 

35. G. P:. Boxer and D. Stetter, Jr., J. Biol. Chem. 155, 237 (1944). 
35a. A. Carlson, AcUt Pfuj-siol. Scanrl. 26, 200 (1952). 

36. R. C Thompson, J. Biol. Chcm. 200, 731 (1953). 

36a. G. C. H. Bauer, Acta Physiol. Scand. 31, 334 (1954). 

37. G. He\tesy, The Svedherg Volume p. 45(5. Stockholm (1944). 

3S. I. S. Edelman, a. H. James, II. Baden mikI F. D. Moore, J. Clin. Inr. 23. 
122(1954). 

39. H. Baden and F. D. Moore, J. Clin. Inc. 23, 122 (1954). 

40. L. Singer and W. L. Armstrong, Proc. Soc. Exp. Biol. Med. 76, 229 (1951). 

41. D. L. Buchanan and A. Nakao, J. Biol. Chem. 198, 245 (1952). 

42. H. H. Donaldson, The Rat p. 314. Philadelphia (1915). 

43. M. Blau, H. Spencer, J. Swernov and D. Laszlo, Science 120, 1029 (1954). 

44. G. HEVE.SY, L. Hahn and O. Rebbe, Dan. Biol. Medd. 14, No. 3 (1939). 

45. K. Kjerulf-Jensen, Acta Physiol. Scand. 3, No. 1 (1941). 

46. J. C. AuB, R. O. Evans, L. JT. IIempelman and H. S. Martland, Medicina 
31, 221 (1952). 



Originally published in the Scientific Monlhlij 83, No. 5 (1956) 



25. PATH OF ATOMS THROUGH GENERATIONS 

G. Hevesy 
From the Tnstitut for Research in Organic Chemistry, Stockholm 

The number of atoms inherited from the mother whicii is passed on to 
the next generation depends on three factors: (i) the fraction of the 
total number of atoms of the mother which were passed on to the newly 
born; (ii) the number of inherited maternal atoms that are replaced 
))efore birth of the grandchild by atoms from nutrients; and (iii) the 
number of maternal atoms still present at pregnancy which can be used 
for development of the embryo of the next generation. 

The quantity mentioned first depends essentially on the weight ratio 
between the mother and the newly born. Sodium, chlorine, and sulfur 
are preferred by the body of the newly born, whereas potassium, calcium, 
magnesium, and phosphorus are preferred by the body of the mother. 
The greatest discrepancy from an equilibrium distribution is seen in 
calcium. The concentration of calcium is about 3 times larger in the 
body of the mother than in the body of the newly born. The reason for 
this is the calcium deficiency of the developing skeleton of the newly born. 

The loss of inherited atoms before reproduction of the animal and the 
stability of the still-present, inherited atoms for the development of the 
embryo of the next generation vary decidedly from element to element. 

It has long been recognized that food supplied to the body is not 
only used to supply energy but is also of great importance in the replace- 
ment of used parts of the body. However, only in the last decades has 
interest begun to develop in the quantitative side of the second function 
of the nutrients and in determining the lifetime of molecules and atoms 
in the bodies of animals and plants. 



REPLACEMENT OF SODIUM ATOMS OF THE BODY 

As the first example for the replacement of atoms present in the body 
by atoms absorbed from food, we shall consider the fate of the sodium 
atoms in the organism. If radioactively labelled sodium— for example, 
in sodium chloride — is supplied to the body, the supplied sodium ions 



PATH OF ATOMS THKOUGH GENERATIONS 235 

mix rapidly with the sodium ions circulating in the hody fluid. There- 
fore, the percentage of elimination of radioactive sodium is equal to the 
percentage of elimination of the total amount of circulating and easily 
exchangeable bound sodium in the body. 

Each day about 4 percent of the radioactive sodium present in the 
human body is eliminated (1 — 3). Therefore, the half-life of the sodium 
atoms circulating in the body is about 2 weeks. About 54 grams of sodium 
are present in the extracellular fluid of an 80-kilogram person. To this 
must be added an intracellular amount of about 33 grams. Of the latter, 
about four-fifths is contained in the mineral constituents of the skele- 
ton (4). The intracellular sodium and part of the mineral skeleton sodium 
enters comparatively fast into exchange with the circulating extracellu- 
lar sodium. Two-thirds of the mineral skeleton sodium, about 18 grams, 
is anchored so strongly that it is retained to a large extent throughout 
life (4—7). Of the sodium that is not strongly anchored— a total of 
70 grams (2x10^^ sodium atoms) — not a single atom is present in the 
body after 162 weeks. These atoms are all replaced by sodium atoms 
from the food. 

All the chief ingredients of the body, with the exception of hydrogen, 
nitrogen, sulfur and iron, take part in the build-up of the apatite in the 
bone. The amount of conservation of a part of the individual atoms of an 
(dement in the body for a long or very long time depends foremost on 
1 he factor of how much of this elements enters into the bone apatite 
and is retained there temporarily or finaly. 

The evaluation of the degree and the speed with which atoms of the 
mineral bone skeleton are replaced by atoms of the blood fluid or the 
lymph is therefore of the greatest importance for our problem. 



EXCHANGE OF PHOSPHORUS ATOMS OF THE SKELETON STRUCTURE 

The question of whether and to what degree the atoms of the skeleton 
structure are exchanged with those of the blood and the lymph was 
raised only after radioactive phosphorus became available as an indi- 
cator for phosphorus atoms. Immediately after the discovery of artificial 
radioactivity by Frederic and Irene Joliot-Curie, radioactive' phos- 
phate was produced. This was first used to answer the question of whether 
an exchange takes place between the phosphate ions of the bone and 
those of the blood (8). A few minutes after radioactive phosphate had 
been given to an adult rat it could be traced in the bone structure. 
The content of phosphorus-32 in the bone skeleton increased very rapidl}' 
at first; after 1 hour, however, the increase was much slower. 

The obvious interpretation of these observations was that a rapid 
exchange takes place betw(H'ti the maikcHi phosphate^ ions of the Ihiid 



236 ADVENTURES IN RADIOISOTOPE RESEARCH 

blood and the phosphate ions lying on the surface of the apatite crystals 
of which the bone structure is built. Hand in hand with this exchange 
goes a biological recrystallization of the skeleton. Crystals of the bone 
structure go into solution, and the new crystals, which have been formed 
from the labelled fluid blood, have to be radioactive. The biological 
recrystallization-— that is, the renewal of the bone skeleton— governs 






Fig. 1. Phosphorus-32 absorption by the tibia of an adult rat 5 minutes 

after intravenous injection (left) and 120 minutes after intravenous 

injection (light). ( X 3) [Photos courtesy of C. P. Leblond 

this exchange process nearly completely after a short time. Autoradio- 
graphs taken bj^ Leblond and co-workers (9) illustrate clearly that 
radioactive phosphate is absorbed by the epiphysial plate of the adult 
rat 5 minutes after injection (compare Fig. 1). After 2 hours, the absorp- 
tion is very distinct. 

It follows from the classical investigations of Paneth (10) that the 
ions in the topmost molecular layer fraction of a crystal powder enter 
into an exchange equilibrium with the ions of a surrounding solution. 
This statement holds only for a part of the ions which are located in the 
topmost molecular la^^er of a well-developed crystal surface — of a mine- 
ral, for example. Even if only a small of the topmost molecular layer 
of the bone apatite took part in the exchange proceedings, it would 
be sufficient for the removal of an important part of the phosphorus-32 
from the plasma which was added to the blood fluid. Three percent of the 
bone phosphate, or maybe even more, settles in the topmost molecular 
layer of the bone apatite, and the phosphorus content of the latter is 
about 700 times greater than that of the blood plasma. If after a while 



PATH OF ATOMS THKOKiH GENERATIONS 237 

1 milligram of the phosphato of the bone skeleton shows tlie same radio- 
activity as 1 milligram oi' phosphate of the blood fluid, it could ))e 
concluded thai the total phosphate content of the bone skeleton was 
renewed during the experiment. This, however, is not all the case. The 
experimental conditions for determining the part of the bone skeleton 
which is renewed are very unfavourable. The specific activity of the 
plasma phosphate falls at first very rapidly, later on very slowly, and 
shows fluctuations during the necessarily long duration of the experiment . 
In order to simplify the conditions for the experiment, the specific activity 
(activity per milligram) of the bound phosphate of a rabbit was held 
constant for 50 days by repeated daily injections. 

It followed that about one-third of the soft epiphysial bone skeleton 
was renewed during the duration of the experiment (11). From the 
hard diaphysial bone structure, a considerably smaller fraction is re- 
newed. The exchangeable part of the bone structure of the rabbit has 
therefore to be limited to about one-third. 



FATE OF CALCIUM ATOMS 

In order to be able to pursue the fate of calcium through several 
generations of mice, we had to know what fraction of the calcium ion 
in the bone skeleton of mice is renewed during a lifetime. Numerous 
experiments concerning the calcium metabolism of the skeleton have 
l)een described (12—16). In determining the fraction of the exchange- 
able skeleton calcium, we continued the afore-mentioned experiments, 
and we replaced the very tedious method by another. We did not investi- 
gate the replacement of the inactive bone calcium by labelled calcium 
in the fluid blood. We bred animals that were evenly marked with 
radioactive calcium, calcium-45. We determined then the fraction of 
radioactive calcium ions that left the animal during its lifetime and that 
were replaced by inactive calcium ions from the food. We supplied 
the mother with radioactive calcium of limited activity (less than 0.5 
microcurie) so that no radiation damage to the animal had to be feared. 
In this way we obtained an evenly activated generation of mice, which 
cannot be obtained by any other method. Each member of a single 
generation had the same calcium-45 content within a few percent. 
The mice received radioactive calcium with their food till they were 
grown up. One of the siblings was killed at birth, and the radioactivity 
of its skeleton was determined. The remaining animals were killed at 
other times and examined. It became evident that after more than 
1 year, which repre-sents a considerable part of the life span of a mouse. 
6.7 ± 7.9 percent of the original calcium atoms of the skeleton could 
still be found. Therefore onlv one-third of the bone skeleton is renewed. 



238 



ADVENTURES IN RADIOISOTOPE RESEARCH 



In another series of experiments we examined newly born mice right 
after birth. The other mice were transferred to an inactive foster-mother 
and analyzed at different times in the following 1.5 years. Figure 2 
shows the results of some of these experiments. One should expect that 
during the intense growth of the first week of life a large part of the atoms 
taken over from the mother are disposed of and replaced by those from 



I 



100 



80- 



^ 60 

c 

<u 

■*— 

c 
o 
o 

o 40 
o 



20- 




100 



200 



300 
doys 



400 



500 



600 



Fig. 2. Loss of inlierited calcium atoms during the life of a mouse. 
We determined the total activity of sibUngs whose mother was fed 
calcium-45. The offspring were killed at different times 



food. In the first 50 days, the loss of atoms from the mother is consi- 
derable. However, in the whole life of a mouse it does not amount to 
more than 50 percent. 

We found about 1/300 of the labelled calcium atoms that the mother 
of the second generation received at its own birth, in the newly born 
of the third generation. The loss in calcium atoms of the ances- 
tors before propagation and through limited transmission to the off- 
spring is repeated from generation to generation. It can therefore 
be found by extrapolation that, of the 6 X 10^^ calcium atoms to be 
found in a mouse weighing 30 grams, not one is present in the 
11th generation of its descendants. The loss of labelled calcium 
atoms in the transition from the second to the third generation 
of one strain of mice amounted to 1/200 only. Calcium atoms from 
the mother should no longer be traceable in the 12th generation of 
these animals. 

That the calcium atoms of the ancestors can be traced in such a long 
series of descendants can be ascribed to the fact that about 99 percent 



PATH OF ATOMS TiriiOTOlI (JEXEHAIIOX.S 239 

of the body calcium is located in the skeleton. The skeleton is therefore 
able to preserve an important part of its building materials. However, 
only a fraction of this calcium is available for Ihe structure of the body 
of the descendants. 



FATE OF WATER MOLECULES 

The calcium and phosphorus atoms of the ancestors of the mouse 
(similar conditions should be valid for human beings) are traceable 
in a long series of generations in the descendants. However, the watcn- 
molecules that the mother transmits to the descendants disappear 
during the life of the first generation. 

Shortly after the discovery of heavy water, we were able to determine, 
thanks to the support of H. C. Urey, who discovered this isotope, the 
half-life of water molecules in the human body. We found that this 
amounts to about 10 days in the body (17) for normal water intake. 
The half-life depends on the amount of water consumed. Schloerb 
and his co-workers (18) also found recently a half-life of 10 days with 
a normal daily intake of 2.7 liters. In the case of a rather large daily 
water intake of 12.8 liters, the lifetime fell, however, to 2.5 days. In the 
case of a normal water intake, after 810 days not a single molecule of 
the 2x10^" water molecules originally present remains in the human 
body. 

The half-life of water molecules in rats was determined to be 2.5 
to 3.5 days. In the mouse it should amount to about 2.5 days. After 
340 days, therefore, not a single maternal water molecule is present 
in the mouse. 

Owing to the large part that the element calcium plays in the devel- 
opment of the skeleton, it is preserved best in the descendants. However, 
even of this element, not a single atom of the ancestors is present in the 
11th and 12th generations. This evidence illustrates the independence 
of the hereditary pattern from an atomic share of the forefathers. It is 
well-known that the hereditary pattern depends on the ability of the 
organism to group in atoms, molecules, and higher cellular units in a 
certain manner. A protein composed of 20 different amino acids and 
having a molecular weight of 100,000 is able to appear in more than 
10^270 isomers, as calculated by Staudinger (19). Therefore, incompar- 
ably more types of proteins can exist than the number of water molecules 
(10^6) which are present in the oceans of the world. Since hereditary 
patterns are tied to the reproducibility of individual proteins or nucleo- 
proteins, there is room for new individual hereditary patterns as long 
as the number of human beings has not reached 10^-"" or even a larger 
figure. 



24(1 ADVEXTIRES IK RADIOISOTOPE RESEARCH 

I 

References 

l.G. Hevesy, Acta Pysiol. Scand. 3, 123 (1942). 

2. G. BuRCH, P. Reaser and J. Gronwnich, J. Lab. Clin. Med. 32, 1 169 (1947). 

3. W. SiRi, Isotopic Tracers and Nuclear Radiations. McGraw-Hill, New York 
(1949). 

i. H. Miller et al., in J. E. Johnston, Editor Medical and Physiological Appli- 
cations, Radioisotope Conference, 2nd, Oxford, 1054, Vol. 1. Aoademic Press. 
New York (1954). 

•j.G. C. H. Bauer, Acta Physiol. Scand. 31, 334 (1954). 

•). I. S. Edelman et al., J. Clin. Invest. 23, 122 (1954). 

7.H. Baden and F. D. Moore, Ibid. 23, 122 (1954). 

8. O. Chievitz and G. Hevesy, Nature 136, 754 (1935). 

9.C. P. LeBlond et al., Amer. J. Anat. 86, 289 (1950). 

10. F. Paneth, Z. Elektrochem. 28, 113 (1922). 

11. G. Hevesy, H. Levi, O. H. Rebbe, Biochem. J. 34, 532 (1940). 

12. L. Singer and W. L. Armstrong, Proc. Soc. Exp. Biol. Med. 76, 229 (1951). 

13. A. Carlson, Acta Phai'macol. Toxicol. 7, Suppl. A (1951). 

14. R. Amprino and A. Engstrom, Acta Anat. 15, 1 (1952). 

15. A. Carlson, Acta Physiol. Scand. 31, 308 (1954). 

16. G. C. H. Bauer and A. Carlson, Ibid. 35, 67 (1955). 

17. G. Hevesy and E. Hofer, Klin. Woch Schr. 13, 1524 (1934). 

18. P. R. ScHOERB et al., J. Chem. Inv. 29, 1926 (1950). 

19. H. Staudinger, Les Prix Nobel (1953), p. 115: Natiirw. Rundschau 9, 8 (1956). 



Originally oommunicated in Acta Chemicfi Scand. 11, 2()1 ( 19.57) 

26. NOTE ON THE CHLORIDE CONTENT OF THE 
MINERAL CONSTITUENTS OF THE SKELETON 

(J. Hevbsy 

From the I)istitnt for Research in Ojganic Chemistry, Stockholm 

In view of the only slightly differing size of the hydroxyl and fluoride 
ion, the hydroxyl ions of the bone apatite can be replaced by fluoride 
ions. Furthermore some of the fluoride may be present in the bone mineral 
as calcium fluoride. 

The fluoride content of the bone apatite is determined by that of the 
plasma, which in turn depends on the fluoride content of the food. 
The fluoride content of the earth's crust is much lower than that of the 
seawater, the mineral constituents of the skeleton of mammals living 
in the sea is, correspondingly, very much, about eleven times, larger 
than that of mammals living on land, which contain about 0.05 % fluoride 
only. The skeleton of fish living in the Baltic, which has a low fluoride 
content, have a much lower fluoride percentage (0.06 %) than the ske- 
leton of fish living in the Atlantic (0.43%). Incorporation of fluoride 
into the mineral constituents of the bone was in recent years much 
investigated, mainly in connection with the observation that the presence 
of fluoride in the mineral constituents of teeth increases their resistance 
to caries. 

The radius of the chloride ion is much larger (1.81 A) than that of 
the fluoride ion (1.33 A) and calcium chloride being very soluble we 
can expect to find slight amounts of chloride in the mineral constituents 
of the bone. While the X-ray diagram of f luoroapatite is almost identical 
with that of hydroxy-apatite that is far from being the case for chloro- 
apatite'^^^ The fluids circulating in the bone tissue having a high chloride 
content (about 300 mgm/100 ml); this has to be quantitatively removed 
prior to the determination of the amount of chloride incorporated into 
the mineral constituents. Such removal by chemical treatment of the 
bone encounters great difficulties. However, when labelling the skeleton 
all through with radiochloride and placing the animal, e. g. the mouse, 
on diet containing non-radioactive chloride for several months, all 
exchangeable radiochloride will be removed and excreted. Ihe radio- 
chloride fixed in the mineral constituents alone remaining in liie ske- 
leton. 

1 6 Hovesy 



242 ADVENTURES IX RADIOISOTOPE RESEARCH 

If the human hali' of the exchangeable chloride is removed in the 
course of 14 days^^) and replaced by chloride of the food and after 6 
months, i.e. after 12 periods, all exchangeable chloride initially present 
will be practically absent. In the mouse, with its high metabolic rate, 
the removal rate of chloride can be expected to be still higher than in 
man. The removal can be accelerated by increased chloride feeding. 
Besides the chloride present in the standard biscuits fed we added 
0.2% NaC'l to the water the mice were drinking after they were put 
on non-radioactive diet. To the water administered to the mice in the 
first phase of the experiment ^^Cl of 0.67 ju C activity per liter was added 
as sodium chloride weighing 9.3 mgm. The labelled chloride was adminis- 
tered to pregnant mice about 2 weeks prior to gestation. After gestation 
the administration of labelled chloride was continued for 4 months 
when the animals were fully grown. One member of each litter was then 
killed and its total ^^C'l content and that of its skeleton determined. 
The remaining members of the litters were investigated 6 months later. 

The bone was ashed in the presence of sodium carbonate. The activity 
of 100 mgm, thus of an infinite thick layer of the samples obtained was 
determined, the counts registered being multiplied by the total weight 
of the ash-sodium carbonate mixture. 

The total ash of the first member of the litter of the mice investigated 
had a total activity of 2780 counts per min, mean value 2910 i 452 
counts, that of the the mineral constituens of the skeleton of the first 
investigated offspring 56.6 counts. This was prior to biological removal 
of all exchangeable chloride from the skeleton but after the removal 
of some of the latter in the course of the isolation of the mineral con- 
stituents. 

By keeping the mice on an activity-free diet for 6 months the activity 
of the mineral constituents declined in the average to 21.2 counts 
(cf. Table 1). 

The total chloride content of a 35 gm mouse taken to be 48 mgm, the acti- 
vity of 1 mgm of the body chloride prior to removal of the active food 
was 60.6 counts per min. As the total skeleton after biological removal 
of all exchangeable chloride had an activity of 21.2 counts per min. 
the sequestered chloride content of the bone mineral amounted to 
0.35 mgm or 73% of the total body chloride. 

The sequestered fraction of the bone calcium of the mouse, that non- 
replaceable by circulating calcium, was found^^) to be 67.2 ± 7.9%. 
The corresponding figure for bone sodium is stated to be 60 — 70('^>, 
65(4), 60<5) and 69(6) by different authors. Thus about a similar percentage 
of excess sodium and of excess calcium is prevented from interchanging 
with their circulating atoms. The sequestration of bone constituents 
is iresumably due to the fact that a contact between these constituents 
and the circulating body fluids is obstructed. To arrive at the total 



CHLORIDE CONTENT OF THE MINERAL CONSTITFENTS OF THE SKELETON 243 

excess chloride content of lh(^ l)one of 1lic mouse we correspondingly 
have to multiply the figure of 0.35 mgm found for the non-exchangeable 
bone chloride by about 1.5, thus arriving at the result that the total ex- 
cess bone cliloricUM-onl (Mil ofour35gm mice aniounls 1o aV)Ou1 0.53 mgm. 

Table 1. — Counts per Min. ^*C1 
Activity of the Skeleton of 33^ — 36 g 
Mice after Being Kept on Non-active 
Diet, thus after Biological removal 
OF Exchangeable RAniocHLORinR, 
FDR () Months 





18.2 




24.(5 




25.1 




28.6 




25.1 




26.6 




25.5 




16.4 




19.4 




12.1 




211.6 : 10 = 21.2 ± 2.08 




m.eV.: = ± 0.208 



In contrast to the bone sodium which is to a large extent present as 
excess sodium in the skeleton it can be shown that the chloride present 
as excess chloride makes out 1/10 only of the total bone chloride. 

From 234 m-equiv. sodium present in 1 kgm of dry human bone 84.9%, 
thus 46 gm, was found by Edelman et alS^'^ to be excess sodium in a 70 kgm 
man. For the dog Edelman and associates found 89.5% of the bone sodium 
to be excess sodium and a similar figure is stated by Miller and associates^"). 
1 kgm of rat bone was found to contain 125 m-equiv excess sodium^^). 
As to the total chloride content of 1 kgm fat free bone this was fountl 
to amount to 19 m-equiv. only(^), thus to less than the extracellular bone 
sodium which makes out 25 m-equiv. While excess bone sodium is to a 
marked extent responsible for the difference between total and extra- 
cellular body sodium, for chloride this difference is almost entirely due 
to the presence of intracellular chloride in Ihc soft tissues. 

References 

1. C. T. Ray, G. E. Burch and S. A. Threfoot, J. Lab. Clin. Med. 39, 673 (1952). 

2. G. Hevesy, Kgl. Danske Videnskab. Selnkab Biol. Medd. 22, Xo. 0. (1955). 
3.G. C. H. Bauer, Acta Physiol. Scand. 31, 334 (1954). 

16* 



244 ADVEXTURES IX RADIOISOTOPE RESEARCH 

4. R. E. Davie.s, H. L. Kornberg and G. AI. Wilson, Biochim. ct BiGp/iys-. 
Acta 9, 403 (1952). 

5. J. S. Edelman, a. H. James, H. Baden and F. D. Moore, J. Clin. Invest. 
33, 122 (1954). 

G. W. H. Bergstrom, J. Clin. Invest. 34, 997 (1955). 

7. H. Miller, D. S. Munro, E. Renschler and G. M. Wilson, Radioisotope 
Conference, Oxford, Vol. I, p. 138. 

8. W. H. Bergstrom and W. M. Wallace, J. Clin. Invest. 33, 867 (1954). 

9. R. Wallacys and G. Chandron, Comt. rend. 230, 1867 (1950). 



245 



Comment on papers 24, 25, 20 

The availability of ■'^Ca at a later date much facilitated the investigations of 
processes taking place in the bone apatite. Experiments taking years could be 
(tarried out, which had not been possible before. Furthermore, in contrast to the 
body phosphorus, which is only partly found in the skeleton, 99 per cent of the 
body calcium of the mouse is concentrated in the latter. In paper 24 experiments 
are described in which ^^Ca was administered to pregnant mice and also to th(^ 
offsprings until they were fuUy grown. After that date, they were kept on a non-ra- 
dioactive diet. Mice were then killed at intervals, and the *^Ca content of their skeleton 
determined. In the coui-se of 2 years, which covers the largest part of the life span of 
the mouse, 33 per cent of the skeleton ^^Ca, and thus of the skeleton calcium, was 
found to be replaced by calcium atoms of the food or present formerlj'^ in the soft 
tissue. When ^^Ca was administered to the pregnant mouse alone, the offspring were 
found to conserve 50 per cent of the maternal calcium atoms through life. When 
similar experiments wore carried out with ^-P (paper 20) 60 per cent of the ^^P 
acquired by the mouse at birth was found to be lost when reaching maturity 
(paper 23). The calcium atoms have a fairly stable position in the skeleton so 
that it takes a great number of generations until the last calcium atom originating 
from the first ancestor is lost. In contrast with calcium atoms, the last ancestoral 
water molecule is lost already after a few generations, as described in paper 25. 
The above-mentioned renewal figures indicate replacement of skeleton calcium 
atoms by such taken up with the food or formerly located in soft tissues. Changes 
in the apatite crystals without participation of food calcium or soft tissue calcium 
would not be indicated by the methods described. 

In the course of a symposium which took place in the Ciba Foundation in 1951 , 
the question was raised of how much chloride is to be found in the mineral consti- 
tuents of the skeleton. This question, which could not then be answered, induced 
an investigation the results of which are found stated in paper 26. The non-exchange- 
able chloride fraction was found to remain very much behind the non-exchange- 
able sodium fraction of the mineral constituents of the bone. 



Originally published in Scand. Arch. Physiol. 77, 148 (1937) 

27. THE FORMATION OF PHOSPHATIDES IN THE BRAIN 
TISSUE OF ADULT ANIMALS 

L. Hahn and G. Hevesy 
From the Institute of Theoretical Physics, University of Copenhagen 

It is generally assumed that no regeneration of the brain tissue of adull 
animals takes place. To test the validity of this assumption we investi- 
gated whether any formation of phosphatides takes place in the brain 
tissue of adult animals. This problem cannot l)e attacked by ordinary 
chemical methods because these do not permit the making of a distinction 
between phosphatide molecules formed at different dates; this is, how- 
ever, possible if we introduce a labelled phosphate into the animal body 
(Chievitz and Hevesy, 1935, 1937), labelled sodium phosphate for 
example, and investigate whether the formation of labelled phosphatides 
can be established in the brain of the animal. We carried out experi- 
ments on rats, mice^ and rabbits. 

Labelled phosphorus can be obtained by adding radioactive phos- 
phorus to ''normal" phosphorus. If we dissolve for example 1000 rela- 
tive (radioactive) units of the radioactive phosphorus isotope ^^P in a 
solution containing 1 mgm of phosphorus, say as sodium phosphate, 
and administer this solution to an animal, then the presence of 1 relative 
radioactive unit in a part of the animal tissue will prove the presence 
of ^/looo ^^ ^^^^ total number of phosphorus atoms administered. The 
radioactive ^^P used in our experiments was obtained by bombarding 
carbon disulphide with fast neutrons from a mixture of radium sulphate 
and beryllium. 

Phosphatides are the second most al)undant constituents of the brain 
tissue. The composition of the latter is shown in Table 1. 



Table 1. — Compositiox of the dky Brain 
Tissue of Adilt Albino Rats 






Protein 

PliDsphatide.s 

Lipides not containing P 1 

.Kstei-s and inorganic constituents . . . 9.S 



48..-) 

2(i.r) 



FORMATION- OF PHOSPHATIDES IX THE BKAIX TISSUE OF ADILT ANIMALS 24 7 

The total phosphorus present, amounting lo 1.39%, is distributed 
between protein, phosphatides and acid soluble compounds in tiie iollow- 
ing manner. 

Protein P 6.8% 

Phosphatide P 67.6% 

Acid soluble P 25.6%, 

the largest amount of phosphorus being thus present as phosphatide. 
Let us assume that labelled sodium phosphate is introduced per os or 
by injection into the animal body and that after the lapse of some 
time some of the inorganic phosphorus present in the brain tissu(> is 
found to be labelled. Such an observation is not to be interpreted as 
a formation of new brain tissue because the inactive phosphate ions 
can be replaced by labelled ones through a simple exchange process. 
The phosphate present in the lecithin molecule cannot, however, be 
replaced through a simple exchange process i.e. the labelled phos- 
phate can only enter the lecithin molecule during a synthesis of the 
latter. The presence of labelled lecithin molecules in the brain is there- 
fore a proof that a synthesis of lecithin has taken place after the intro- 
duction of the labelled sodium phosphate into the animal body. Though 
it was highly improbable that the phosphate present in the lecithin- 
molecule could be replaced by labelled phosphate through a physical 
exchange process, we tested this point by carrying out the following 
experiment. We shook 10 cc. of cats blood at 37" for 4 hours with 
2.5 cc. of isotonic sodium chloride solution containing labelled phos- 
phorus. 10 cc. of blood contain about 1.2 mgm of phosphatide and 
0.4 mgm of inorganic phosphorus, the latter being labelled by the 
activity added. The lecithin was then extracted. The extraction was 
made with ether -f- alcohol and the extract was shaken for several 
hours with calcium phosphate to remove any inorganic labelled 
phosphate which might be present in the extract. Of the 10,000 
radioactive units only 3 units were found in the blood phosphatide 
extracted. Even this very slight exchange is probably due to en- 
zymatic actions occurring in the blood. From an experimental point of 
view blood seemed to be a very suitable liquid to carry out an exchange 
experiment and as the exchange observed was only a very slight 
one it did not seem of interest to pursue the subject further and carry 
out exchange experiments in liquids from which enzymes had been 
removed. In this connection we may, however, mention an experiment 
in which blood containing labelled phosphate was allowed to circulate 
through an isolated liver. 

Professor Lundsgaard being engaged on liver perfusion experi- 
ments kindly added a solution (3 cc.) containing labeUed phosphorus 
of negligible weight to the blood used in his experiments. In 10 cc. 



248 ADVEXTURES IN RADIOISOTOPE RESEARCH 

of blood used for 4 hours, 7 of the 10,000 radioactive units added were 
found in the isolated lecithin phosphorus; the liver perfusion has thus 
a positive effect on the formation of labelled lecithin. 

We may also mention that in 1 cc. of the blood of a cat killed li.> 
hours after injecting a negligible weight of labelled phosphorus, we found 
a lecithin phosphorus activity amounting to 2 % of the activity found 
in 1 cc. of plasma, while the acid soluble phosphorus present in 1 cc. 
of blood corpuscles contained, as Professor Lundsgaard found, an 
activity of 12.5% of that of the plasma, about 2/3 of this being present 
in the phosphorus esters. A detailed study of the distribution of the 
labelled phosphorus between plasma and blood corpuscles is being carried 
out by Professor Lundsgaard and one of the present writers. 



EXTRACTION OF PHOSPHATIDES 

The usual method of extracting phosphatides is by means of alcohol- 
ether mixtures. In this method of extraction a small part of the 
inorganic phosphorus present is dissolved as well; but in view of the 
preponderance of phosphatide phosphorus in the brain the error thus 
introduced can generally be disregarded. Under the peculiar condi- 
tions which prevail in the investigation of the formation of labelled 
phosphatides the error mentioned above can however become very 
embarassing. We introduce labelled inorganic phosphorus into the 
animal body per os or by subcutaneous injection. Now it is possible 
that only a very small amount of this is converted into labelled phos- 
phatide phosphorus so that even of a trace of the labelled inorganic 
phosphorus is extracted by the alcohol-ether mixture, our results can 
be seriously falsified. This is best seen from the following example: 
brain tissue is shaken in vitro with a solution of labelled inorganic 
phosphorus containing 10,000 relative radioactive units, and 0.1 mgm 
phosphorus; the solution is then removed and the dried tissue extracted 
with alcohol -j- ether. The presence of as little as 10~* mgm of in- 
organic phosphorus in the extract corresponds to 10 relative radioactive 
units and may be partly or wholly responsible for the activity of the 
extract. For this reason, although conditions in experiments in vivo 
are much more favourable than those in the example above, we chose 
a method of extraction more suitable for our special case than the alcohol- 
ether extraction. 

The procedure adopted by us was as follows. We dried the brain 
tissue with acetone and extracted the phosphatides by prolonged shaking 
with carefully dried ether; the extract obtained was evaporated to 
dryness and dissolved a second time in ether in the presence of a large 
excess of finely powdered dry sodium phosphate. We had found that 



FOR>L\TION OF PHOSPHATIDES IN THE BRAIN TISSUE OF ADULT ANIMALS 249 

by shaking a solution of laboUed sodium phospluilc wilh a large excess 
of finely powdered unlabell(Ml sodium phosphate the former can ])e 
removed from the solution. A distribution of the phosphate ions between 
the liquid and solid phase takes place and the chance of a lal)elled phos- 
phate ion being in solution is entirely negligible on account of the over- 
whelming excess of the solid phase; this is especially so when the whole 
procedure is repeated. Another method of purification of the ether 
extract fiom the labelled inorganic phosphorus was as follows. Unlabelled 
sodium phosphate was dissolved in the extract and precipitated as 
ammonium magnesium phosphate. By repeating this procedure it was 
possible to get rid of the slightest trace of labelled inorganic phosphorus 
present in the ether extract. The activity of the ether extract had then 
to be measured. The amount of material being small (2.3 mgm) it was 
advisable to add a carrier. An inactive commercial lecithin preparation 
was used for this purpose, some of it being dissolved in the ethereal 
solution before evaporation. Before destroying the lecithin, calcium 
oxide was added to bring about the formation of calcium phosphate. 
The activity of the latter was measured by means of a Geiger— Miiller 
Counter. 



INVESTIGATION OF THE BRAIN OF RATS 

All our investigations were carried out on fully-grown adult rats- 
One animal was killed after a lapse of 5 days, a second one after a lapse 
of 3 days, and the third one after one hour. The results obtained are 
seen in Table 2. 

Table 2 





Fresh weiglit 
of brain 
in mgm 


Dry weight 
of brain 
in mgm 


Percentage of labelled P found 


Kut killed after 


in the brain 


in brain 
phosphatide 


5 days 

3 „ 

V24 .. 


1800 
1440 
1430 


380 
300 
290 


7-10-2 

7-10-2 

6.8-10-2 


3.7- 10-2 

2.4-10 2 

0.42-10-2 



The above figures show clearly the formation of labelled phosphatide 
in the brain of adult animals. Though it could hardly be doubted that 
what we extracted and tested was actually phosphatide we obtained 
further evidence of this preparing from the brain tissue of rats to which 
labelled phosphorus had been administered, the highly characteristic 
chlorocadmium compound of lecithin and tested its activity. 



250 



ADVEXTURES IX RADIOISOTOPE RESEARCH 



PREPARATION OF CHLOROCADMIUM LECITHIN 

The ether extract of the brain was evaporated to dryness, the residue 
was dissolved in alcohol, and a saturated solution of cadmium chloride 
in methyl alcohol added. The chlorocadmium lecithin which precipitates 
from the solution was further purified in the following manner : the 
precipitate was suspended in chloroform and a solution of ammonia 
in methyl alcohol added, whereupon the lecithin remained in solution 
while the cadmium is precipitated. The next step was to evaporate 
down the solution containing the lecithin, and to dissolve the latter 
in alcohol; the whole purification process was then repeated. We ascer- 
tained in our preliminary experiments that the above lenghty process 
can be carried out with a yield of about 50%. In an experiment with 
a rat's brain extract which showed an activity of 2.4 -lO'^ o^^ of the 
total amount given to the animal, we recovered, after the preparation 
of the chlorocadmium compound, lecithin containing 1.2 •lO"^ o^^ 
of the total activity administered. 



INVESTIGATION OF THE BRAIN OF MICE 

As has been mentioned already it is of great importance to investigate 
the brains of animals no longer growing. Through the kindness of Pro- 
fessor Krogh and Dr. Hagedorn we obtained mice of unusually high 
age and investigated their brain. The animal was killed 21 days after 
injecting the labelled phosphorus. A result obtained was the following: 

Table 3 



Weight of fresh 


Weight rif 


Percentage of labelled phos- 
phorus administered found 


J 


in the brain 


in brain lecithin 


814 mgm 


86.2 mgm 


1.3.5- 10-1 


.5.5-10-2 



INVESTIGATION OF THE BRAIN OF RABBITS 

The investigation of a brain of a rabbit killed 27 days (I.), resp. 4.5 
hours (II.) after injection with radioactive phosphorus gave the follow- 
ing result. 

Table 4 



Weight of 
fresh liruiii 


Weight of 
dry brain 


Percentage of labelled phos- 
phorus administered found 


in the brain . 


in brain lecithin 


I 8940 mgm 
II 


1400 mgm 


6.9-10-2 
1.8-10-3 


2.3-10-2 
6.3-10-3 



FORMATION OF PHOSrHATIDES IX THE BI5AIN TISSTK OF ADULT ANIMALS 251 

RELATIVE ABUNDANCE OF LABELLED PHOSPHORUS IN THE BR4IN 
COMPARED WITH THE AMOUNT PRESENT IN OTHER ORGANS 

The relative abundance of labelled phospliorus in different organs 
(activity per mgm phospliorus) is seen from Table 5. 

Tahlk .") 



Aiiiinal 



K'iUiMl after 



Total Bi-aii 
(A) 



liraiii 

liecithiu 

(B) 



Bone 



Rat 5 days 

Rat 3 days 

Rat 1 hour 

Mouse j 21 days 

Rabbit I 27 days 



2.12 
2.12 
2.12 

0.94 
fi.lO 



2.34 
2.10 
0.40 
1.08 
6.10 



0.9 
1.0 
.5.3 
0.9 
1.0 



1 (tibial 

1 

1 „ 

1 ,r 

1—3.5* 



* 1 = Tibia diaphysis, 2.6 = Jaw, 3.5 = Tibia epipliysis. 

As is seen from the above figures the ratio between labelled phosphorus 
in the whole brain and labelled phosphorus in brain lecithin shifts in 
favour of the latter with increasing time; furthermore that the percent- 
age replacement of phosphorus atoms in the brain by labelled phosphorus 
atoms is not very different from that found in the case of the phosphorus 
atoms of the bone. 

Note added in proof : Professors Artom, Perrier, Sarsana. 
Santagello and Segre most kindly sent us a manuscript of a paper 
in which the formation of lipidic phosphorus in different organs has 
been demonstrated by using radioactive phosphorus as indicator. They 
found the most marked metabolism in the liver, the intestinal mucosa 
and the kidney, the least in the brain and the muscles. 



WEIGHT OF THE NE\> LY FORMED LECITHIN 



So far we have only calcula1(>d lecithin phosphorus formed as a per- 
centage fraction of the total activity given to the animal. In what 
follows, we will try to calculate the amount of newly formed labelled 
lecithin in grams. This is a more difficult problem. If we inject 1 mgm 
of phosphorus showing an activity of 1000 units we know that lh(> 
presence of 1 unit of activity indicates Vioon "^g"^ o^ ^^^^ phosphorus 
atoms injected. Now in the blood plasma of a rat we have for 17 cc. 



252 ADVENTURES IN RADIOISOTOPE RBSBARiClH 

of blood with an inorganic phosphorus content of 4.5 mgm per 100 
cc, 0.75 mgm of phosphorus; if we inject radioactive phosphorus 
(1000 units) of negligible weight, 1 activity unit will indicate 0.75/1000 
mgm of the phosphorus originally present in the plasma. In the 
blood plasma, however, numerous fast processes take place in which 
the phosphorus atoms are involved; this is shown by investigations 
carried out with labelled P. We get beside other reactions, a partition 
of the phosphate ions between the plasma and the phosphate in the 
skeleton, and in view of the very large preponderance of the latter a large 
part of the labelled phosphate ions will soon be present in the skeleton. 
Sim.ilar considerations apply to the muscles and other organs. The com- 
paratively large amount of phosphorus ester present in the blood cor- 
puscles will also take part in a dynamic interchange with the labelled 
phosphorus atoms present in the plasma. In the course of the last two 
years Prof. E. Lundsgaard and one of us have carried out and extended 
investigation of this point, to be published shortly. As a result of the 
processes just mentioned, only a few, let us say 10, units remain behind 
of the 1000 activity units introduced into the plasma, while the total 
inorganic phosphorus content of the plasma is unchanged and amounts 
as before to 0.75 mgm. The effect will be that 1 activity unit will now 
indicate as much as 0.75/10 mgm (i.e. much more) phosphorus. On 
account of the rapid dynamic happenings in the animal body our scale 
of indication will rapidly change and, the function representing this 
change being a very intricate one. One way to obtain some infor- 
mation is the following. We make an experiment of very short du- 
ration in which 1 activity unit does actually indicate 0.75/1000 
mgm of labelled phosphorus; then we make a longer experiment and 
determine experimentally the activity of the inorganic phosphorus 
present in the plasma. If only 10 activity units are now present then 
1 activity unit indicates 0.75/10 mgm P at the latter stage. The activity 
accumulated at the early stage will be correctly interpreted by making 
use of the first mentioned scale, that accumulated at the last stage 
by making use of the last mentioned scale. In some cases we have followed 
the labelled P content of the blood continuously. Let us now consider 
the rat killed after 1 hour, the blood plasma of the animal containing 
0.75 mgm inorganic phosphorus, a 4.2 • lO^^th part i.e. 3.2 • 10"^ mgm of 
this was converted into brain lecithin phosphorus. This being a very 
small part of the brain lecithin phosphorus, the reverse reaction can bo 
disregarded and the amount of labelled brain lecithin phosphorus 
formed in 100 hours can be taken as a hundred times as much, i.e. 3.2- 10"^ 
mgm i.e. 8 mgm of the brain lecithin is newly formed in the course 
of about 4 clays. The calculation of the weight of labelled phosphorus 
was carried out on the basis of an analysis of the activity of the blood 
and of the brain lecithin after the lapse of one hour. Now one part of 



FORMATION OF PHOSPHATIDES IN THE BRAIX TISSUE OF ADILT ANIMALS 25!^ 



the labelled lecithin was already formed after the lapse of a few minutes, 

, . , . , ,. mass of inorganic P . , , , , , 

at which time the ratio in the blood was smaller 

activity 

and 1 activity unit therefore indicated a smaller mass of phosphorus 

than after the lapse of one hour when the measurements were actually 

made. Hence the actual amount of labelled lecithin phosphorus will 

be less than that calculated above but is definitely higher than 0.08 mgm. 



PHOSPHORUS EXCHANGE IN BRAIN LECITHIN IN VITRO 

We shook a freshly removed rat brain for 5 hours at 37° with 3 cc. 
ol' an isotonic sodium chloride solution containing 0.09 mgm of phos- 
phorus labelled by the addition of radioactive phosphorus. The brain 
was carefully washed with cold acetone and dried at room tempera- 
ture and the lecithin extracted as described above. The ethereal solu- 
tion was shaken for several hours with sodium phosphate to remove 
inorganic labelled phosphorus and after this operation had been repeat- 
ed four times, the sodium phosphate was found to be entirely inac- 
tive. The solution showed an activity of 2200 units and we found 18 
units in the lecithin extract; this corresponds to 0.00076 mgm of label- 
led phosphorus or about ^I^qqq part of the total lecithin phosphorus 
content of the brain. Thus the formation of a very small amount of 
labelled phosphorus also takes place in the freshly removed brain tissue 
in vitro, presumably under enzymatic action. We intend to follow up 
the exchange problem ii} vitro in greater detail. 



DISCUSSION OF THE RESULTS 

From the results above it clearly follows that brain lecithin is con- 
stantly being synthesized in the brain tissue of adult animals. Pre- 
sumably a part of the lecithin is constantly broken down under enzy- 
matic action and rebuilt again, thus making it possible for the labelled 
phosphorus atoms present in the blood to enter the lecithin molecule. 

Belfanti, Contardi and Ercoli (1936) give the following scheme 
according to which lecithin is supposed to decompose under the action 
of lecithases. 

It is possible that a reaction takes place in both directions according 
to this or a similar scheme, so that the phosphorus atoms present in 
lecithin, are rendered exchangeable when enzymes are present although 
they undergo no exchange in the absence of these. The study of the 
mode and rate of action of the different lecithases may be much facili- 
tated by following up exchange process in lecithin and its decomposition 
])roducts in 1ho prosonoo and absence of the flifferent enzvmes. 



254 



ADVENTURES IX RADIOISOTOPE RESEARCH 




Lecithin 



Fatty acid 

+ 
Lysochitin 




Fatty acids 

Glycerophosphorie ester 
of Cholin 

Cholinphosphatase 

Cholin -f Glycerophos- 
phorie acid 

Glycerophosphatase 
Glycerin + Phosphoric acid 



The figures in our experiments show clearly that the ratio of labelled 
lecithin phosphorus to labelled phosphorus other than that from lecithin 
in the brain increases with time. The amount of labelled lecithin produced 
within 1 hour in the brain of a rat can be estimated as lying between 
0.08 and 0.0008 mgm. 

In starting this research we contemplated the possibility of the for- 
mation of labelled brain lecithin being influenced by nervous action. 
No effect of nervous actions on chemical processes in the brain has 
yet been ascertained; the dependence of the latter on the former must 
be described by a curve where a very large increase in the abscissa 
(nervous action) corresponds to a minimal change in the ordinate 
(chemical effect). To test this possible effect of nervous action it 
would be necessary to carry out a very large number of experiments. 



Summary 

By using laVjclled (radioactive) phosphorus as indicator, it was found that one 
hour after the subcutaneous injection of labelled sodium phosphate, labelled 
lecithin was already formed in the brain tissue of fully grown rats. Similar experi- 
ments were also carried out with fully grown mice and rabbits. The losult proves 
that a constant breakdown and building up of lecithin takes place in the brain 
tissue presumably under enzymatic action. 



References 

O. Chievitz and G. Hevesy (1935) Nature 136, 754. 

O. Chievitz and G. Hevesy (1934) Kgl. Danske Vidensk. Sehk. Biol. Medd. 

XIII, 9. 
S. Belfakti, a. Contardi and A. Ercoli (1936) Ergehn. Enzymforsch. 5, 213. 
C. Artom, L. Perkier, M. Santagello, G. Sarzana and E. Segre (1934) Nature 

139, 83G. 



Originally publishod in Xaturc, 140, 275 (1!)37) 

28. LECITHINAEMIA FOLLOWING THE 
ADMINISTRATION OF FAT 

G. Hevesy and E. Lundsgaard 
From the Institute of Theoietical Physics and the Physiological Insi i1 ute. University 

of Copenhagen 

About two hours after tht^ administration of a meal containing fat. 
the fat content of the blood begins to rise. Bloor^ found that when olive 
oil is administered to a dog, besides an increase in the neutral fat content 
of the blood an increase in its lecithin content also takes place. The 
average increase was found to be about 20 per cent. A maximum is 
reached after four hours. Bloor was inclined to ascribe the lecithin formed 
after the administration and resorption of the neutral fat to a synthesis 
occurring inside the red blood corpuscles. Other explanations might, 
however, be suggested as well, namely: (1) The lecithin is synthesized 
in the intestinal mucose and resorbed into the blood. (2) The synthesis 
takes place, after the resorption of neutral fat, in the liver, or some- 
where else outside the intestinal tract. (3) The increase in the lecithin 
content of the blood is due to mobilization of preformerl lecithin aftci 
the resorption of the neutral fat. 

To decide which of these suggestions is to be accepted we repeated 
Bloor's experiment, but administered simultaneously with the oil- 
labelled (radioactive) phosphorus in the form of sodium phosphate. In 
the case denoted by (1) the additional blood lecithin should contain 
chiefly labelled phosphorus; in case (2) the additional lecithin should 
contain only small amounts of labelled phosphorus; in case (3) the ad- 
ditional lecithin should contain ordinary phosphorus only. 

We determined the normal P present in the blood lecithin, which was 
extracted by the usual procedure, by the method of Fiske and Subbarow, 
and the labelled P by means of a Geiger counter. While, as seen in the 
table, the lecithin phosphorus content of 100 cc. of blood increased by 
2 mgm four hours after administering the oil, that of labelled P only 
increased by 0.096 mgm. We must, furthermore, take into account the 
fact thai half the labelled phosphorus administered two hours before 
Ihe oil pr'oduced 0.028 mgm labelled lecithin P during that time. We 
must ther-efore deduct 2 x 0.028 mgm IVom the 'oil effect" of 0.096 mgm. 
obtaining r).04 niL'^m per 100 cc. ol hlood foi' th(^ maximuni vahie of the 
'oil clfect". 



256 



ADVENTURES IN RADIOISOTOPE RESEARCH 



An important objection can, however, be raised to our conclusion! 
it may be argued that the intestinal tract might contain large amounts 
of phosphorus other than the labelled phosphate administered by us, 
the presence of which must be accounted for when carrying out the 
above calculation. To investigate this point and to ascertain to what 
extent the labelled phosphorus was resorbed, we killed the dog after the 
last experiment, the results of which are seen in the table. We washed 
ihe intestinal tract with water and determined both its total P content 
and its labelled P content. We found by activity measurements 39.6 
mgm labelled P and by chemical determination 175 mgm normal P. 
Within six hours as much as 259.4 mgm of the 300 mgm administered 
to the dog was thus resorbed. The 135 mgm. non-labelled phosphorus 
reached the intestine, presumably along with the digestive fluids, so 
that the 40 mgm labelled P were 'diluted' to 175 mgm. We determined 
also the total acid-soluble phosphorus content of the intestinal mucose; 
it was found to amount to about 40 mgm, bringing the above figures 
up to 215 mgm. But even if we make the assumption that this dilution 
was present during the whole of the resorption process we should get 
the result 5.2 x 0.064 = 0.21 mgm per cent lecithin P. while an in- 
crease of 2 mgm per cent was found in the blood lecithin P. 



Time 




Lecithin phosphorus found 
in 100 cc. blood 


Labelled total P found 


in 


Labelled P given in mgm. 


in 100 cc. blood 




hours 


Total 


Labelled 


in the total blood 
of the dog 





1.50 


„ 


_ 




2 


1.50 (+ 50 gm oil) 


16.0 mgm 0.028 mgm 


1.03 mgm 


G.18 mgm 


4 


— 


15.5 „ 0.048 „ 


2.03 „ 


12.18 „ 


6 


— (259.4 mgm 










resorbed) 


18.0 „ 0.09<) ., 


2.00 „ 


12.00 „ 



It is of interest to compare the labelled P content resorbed with that 
actually found in the l)lood stream of the dog. Six hours after the beginn- 
ing of the experiment, as is seen in the table, only 4.6 per cent of the 
amount resorbed was found. This result illustrates beautifully the great 
rapidity of the phosphorus exchange in the body. As observed by us in 
numerous cases, the individual phosphorus atoms present in the blood 
stream exchange their places rapidly with others present in the different 
organs. For this reason we can conclude with certainty that during our 
experiments the ratio labelled phosphorus to ordinary phosphorus must 
have been appreciably higher in the intestinal mucose than in the blood. 

The only moderate increase in labelled phosphorus in the blood leci- 
1 hin after administration of oil, an increase which nevertheless in all 
our experiments exceeds the increase observed after the radioactive 



LECITHIXAEMIA FOLLOWING THE ADMIXISTRATION OF FAT 257 

phosphorus was adniinislorcd alone, leads to the eonelusion 1 liat during' 
the absorption of neutral lat, lecithin is formed outside the intestinal 
tract. A comparatively rapid formation of labelled lecithin in several 
organs in the course of normal metabolism has in fact recently been 
observed^. 



References 

1. W. R. Bloor, J. Biol. Chem. 23, 314 (1915), 24, 448 (1916). 

2.C. Artom, G. Sarzana, M. Santagello and E. Segre, Nature 139,830(1934), 

Comp. also: 
L. Hahn and G. Heve.sy, Scand. Archiv. f. Phys. (Aug. 1937). 



1 7 Hevesy 



Originally published in Biochem. J. 32, (1938) 

29. FORMATION OF PHOSPHATIDES IN LIVER 
PERFUSION EXPERIMENTS 

L. A. Hahx and G. CH. Hevesy 

From the Institute of Theoretical Physics, University of Copenhagen 

About 2 hr after a meal containing fat, the fat content of the blood 
begins to rise. This alimentary lipaemia is followed by lecithinaemia 
[Reicher, 1911; Bloor, 1915], the phosphatide content of the blood 
increasing more or less parallel with the fat content. A maximum is 
reached after about 4 hr and after 8 hr the fat and phosphatide contents 
of the blood are almost at the initial level. As to the origin of the phos- 
phatides responsible for alimentary lipaemia the following possibilities 
exist: 

(1) the phosphatides are synthesized in the intestinal mucosa and 
resorbed into the blood; 

(2) they are synthesized in the blood; 

(3) they are mobilized under the influx of lipaemic blood from the 
liver or other organs and possibly wholly or partly formed in the former 
during the influx. 

To obtain further information on the above problem, oil together with 
labelled (radioactive) sodium phosphate, were administered to a dog 
[Hevesy anclLuNDSGAARD, 1937]. If the increase in the phosphatide con- 
tent of the blood which amounted to 15 % after 4 hr. was due to phos- 
phatides taken up from the intestine, the phosphatides extracted from 
blood should have shown a marked radioactivity. The latter was, how- 
ever, much smaller than to be expected on this assumption. It follows that 
while phosphatides are synthesized in the intestinal mucosa [Artom 
et al., 1937; Sinclair and »Smith, 1937] and some do enter the circulation 
from the bowels [Himmerich, 1934; Sxillmann and Wilbrandt. 
1934; Freeman and Joy, 1935] the bulk of the phosphatides which 
are responsible for the alimentary lipaemia must originate from outside 
the intestinal tract. We next tested the possibility that the additional 
phosphatides are formed in the lipaemic blood [Hahn and Hevesy. 
1938]. A few ml. of dog blood were shaken with labelled sodium phos- 
phate under the usual precautions for 4.5 hr. The phosphatides ex- 
tracted after the experiment were only slightly radioactive, the labelled 



FORMATIOX OF PHOSPHATIDES IX LIVER I'EKFLSION EXFEIU.MEXTS 



259 



phosphatides formed amounting to only about 0.1% of tlie total amount 
present. No differenee was found in the behaviour of normal and lipaemic 
bloods. 

FORMATION OF LABELLED PHOSPHATIDES IN PERFUSION 

EXPERIMENTS 

Through the great kindness of Prof. Lundsgaard and Dr Blixen- 
CRONE, who carried out perfusion experiments on isolated livers, we were 
enabled to test the formation of labelled phosphatides in the blood of 
cats circulating through an isolated liver and also in the liver tissue. 
To 120—160 ml. of cat blood diluted to about twice its volume with 
physiological NaCl solution a minute amount of active sodium phos- 
phate was added. The blood was then defibrinated and allowed to circu- 
late through an isolated liver for 2.5 hr. The labelled inorganic P present in 
the blood was determined at the start and at the end of the experiment 
and also the labelled phosphatide P of blood and liver at the end of 
the experiment. The ratio of the specific activity (activity per mgm P) 
of the blood phosphatide P to that of the blood inorganic P is seen from 
Table 1. In interpreting the figures of the table we should recall that 
if all phosphatides molecules present are newly formed the specific 
activities of the inorganic P and phosphatide P should be equal. The 

Table 1 
Specific activity of blood phosphatide P 

Specific activity of blood inorganic P 



(average value) 



Normal blood (av. of 3 exps.) 
Lipaemic blood (av. of 2 exps.) 



0..5 X 10^3 
1.6 X 10^3 



1.2 X 10^3 
3.4 X 10~3 



0.85 X 10~3 
2.5 X 10-3 



figure of 0.85 xlO"^ for the ratio quoted, for example, shows that the 
new formation of phosphatide molecules within the experiment amounts 
to only 0.085%. In the course of the experiment inactive inorganic P 
of the liver and also a part of the P present in the organic P compounds 
of the liver exchange with the active plasma inorganic P and lower the 
specific activity of the latter. The specific activities of the plasma inor- 
ganic P being thus different at the start and at the end of the experiment 
we have calculated the ratio (seen in Table 1) for the beginning of the 
experiment (col. 1), for the end (col. 2), and also an average value (col. 3). 
The figures of Table 1 for normal blood hardly differ from the figures 
obtained in the experiments in vitro (average value 0.8x10"^). While, 



17* 



260 



ADVENTURES IN RADIOISOTOPE RESEARCH 



however, in the experiments in vitro no definite difference was found 
in the formation of phosphatides in normal and lipaemic bloods, in the 
perfusion experiment about three times as many newly formed phospha- 
tide molecules were found to be present in the lipaemic blood as in the 
normal. This result is supported by figures obtained when investigating 
the labelled phosphatides extracted from the livers used in the perfusion 
experiments. Here also (see Table 2) a greater part of the phosphatide 
present became labelled when lipaemic blood was used. 



Tablk 2 









Spec, activity of 


Liver 


Phosphatide P 

pergm £resh tissue 

mgm 


Total P per gm 
fresh tissue mgm 


phospliatide 
P X 100 




Spec, activity of 








inorganic P 




Perfusion with normal 
blood 




1 


0.96 . 


2.G 


1.2.3 


2 


' 1.13 


2.9 


1..53 


3 


0.88 


2.0 


1.78 




Perfusion with lipaemic 






blood 


, 


4 


0.7.1 


2.4 


2.75 


f) 


0.6'J 


2.2 


2.59 


6 


1.32 


3.7 


2.79 



The livers used were taken from fasting cats, except in Exp. 3. In 
Exp. 6 the specific activities of the ester P of the liver and the protein 
P (remaining P after extraction with ether-alcohol and trichloroacetic 
acid) were determined as well. The relative figures ol)tained were; speci- 
fic activity of the inorganic P, 1; of the ester P, 0.218; of the protein 
P, 0.068. The radioactive P atoms can only enter into the phosphatide 
molecules by a synthetic process. If the radioactivity of 1 mgm organic 
P of the liver were equal to that of 1 mgm inorganic P, all organic P atoms 
would have been replaced. If the organic P were not radioactive at all, 
none of the organic molecules could be newly formed. If all the phospha- 
tide molecules present in the liver after the perfusion experiment had 
been newly formed the value of the ratio in the last column would be 1. 
From the figures it follows that 1.5% of all phosphatide molecules pre- 
sent in the experiment with normal blood and 2.7% in that with lipaemic 
})lood are formed in the course of the experiment. 

During the perfusion experiment some molecules may have decom- 
posed thus introducing an uncertainty into all conclusions based on the 
determination of the amount of phosphatides present. Our conclusions 



FORMATION OF IMIOSPHATIDES IN LIVER PERFUSION EXPERIMENTS 2G1 

are not influenced, however, by this source of error, since they are not 
based on determinations of the phosphatide content before and after 
perfusion but on the ralio of hiliclled and non-labelled phosphatide 
molecules present in the liver. We may, therefore, conclude from the 
results described above that lipaemic blood is more effective in the 
formation of phosphatides in the liver than is normal blood. This result 
suggests that one of the main reasons for alimentary lecithinaemia is that 
during the influx of lipaemic blood phosphatide formation in the liver 
is increased and phosphatides are discharged into the circulation [cf. 
Aylward et al., 1935]. As the lipaemic blood is changed into normal 
blood the excess phosphatides are taken up by the liver and other 
organs, until the "normal" phosphatide content of the blood is reached. 



Suiiiniary 

In oxporimonls in vitro llio amount of labollod phosphatido obtained in shak- 
ing blood with radioactive sodium phosphate is the same whether normal or 
lipaemic blood is used. 

In perfusion experiments the lipaemic blood is found to contain more labelled 
and thus newly formed phosphatide than the normal blood. The same result 
applies also to the phosphatides extracted from the liver in the perfusion experi- 
ment. 



References 

Artom, Sarzana, Santageixo and Segre (1937) Nature 139, 836. 

Ayl Ward, Channon and Wilkinson (1935) Biochem. J. 29, 172. 

Bloob (1915) J. biol. Chern. 23, 317. 

Freeman and Joy (1935) J. biol. Chern. 114, 132. 

Hahn and Hevesy (1938) Mem. Carlsberg Lah. 22, 188. 

Hevesy and Lundsgaard (1937) Nature 140, 275. 

HiMMERiCH (1934) Amer. J. Physiol. 116, 342. 

Reicher (1911) Verh. Kongr. inn. Med. 28, 327. 

Sinclair and Smith (1937) J. biol. Chern. 121, 361. 

SxtivLMANN and Wilbrandt (1934) Biochem. Z. 270, 52. 



Originally communicated in Kgl. Danske Videnskabernes Selskab. Biologiske 

Meddelelser, 15, (1940) 

30. RATE OF PENETRATION OF PHOSPHATIDES 
THROUGH THE CAPILLARY WALL 

G. Hevesy and L. Hahn 
From the Institute of Theoretical Physics, University of Copenhagen 

Ions or molecules of crystalline substances present in the plasma can 
easily penetrate through the capillary wall. As soon as a few minutes 
after injecting labelled sodium ions (^^Na^) into the jugularis, we find 
these ions proportionally distributed between the sodium ('^3]s^a~^) ions 
of the plasma and those of the interspaces. On the other hand, colloidal 
particles like those formed by the proteins of the plasma under physiolo- 
gical conditions pass through the walls of the capillaries at very slow 
rate only. The phosphatides present in the plasma can be expected to 
have an intermediary position as to their penetrability through the 
capillary wall between the crystalline constituents and the proteins 
present in the plasma. To determine the rate of penetration of the plasma 
phosphatides through the capillary wall, we introduced labelled phos- 
phatides (phosphatides containing radioactive P) into the plasma and 
measured the rate of their disappearance from the circulation. 

The labelled phosphatides were obtained in the following way. Label- 
led sodium phosphate was administered to a rabbit (A). The phosphati- 
des formed, after the start of the experiment, in the liver and other 
grgans of this rabbit become labelled ; a part of these labelled phospha- 
tides is liberated into the plasma. By injecting plasma of this ral:)bit 
(A) into the circulation of another rabbit (B), we introduced labelled 
plasma phosphatides under strictly physiological conditions into the 
circulation. To avoid the increase of the plasma volume of rabbit B, 
we removed, previous to the injection of the labelled plasma, for example, 
20 cc. blood of rabbit B. This blood was, after addition of heparin, 
gently centrifuged to separate the bulk of its plasma content which 
was then replaced by the labelled plasma of rabbit A. The blood thus 
obtained was injected into the jugularis of rabbit B. This rabbit, thus, 
gets its own corpuscles reincorporated, combined with the corresponding 
amount of labelled plasma of the other rabbit. An aliquot part of the 
plasma of rabbit A is kept to be analysed. 

The labelled phosphatide molecules introduced into the circulation 
of rabbit B become distributed in the total plasma of the rabbit almost 



RATE OF PENETRATIOX OF PHOSPHATIDES THROUGH THE CAPILLARY WALL 203 

at once, the next step being the continuous escape of the labelled phos- 
phatide molecules through the capillary wall and their replacement 
by other phosphatide molecules, originally located in the organs, which 
diffuse in the opposite direction, namely through the capillary wall, 
into the plasma. Since the phosphatide content of the plasma remains 
practically constant during the experiment, the exodus of a certain 
quantity of phosphatides must be followed by the influx of about th(> 
same amount. Tn view of th(> very minute turnover of ])ho,sphatiflos 



O = first experiment 
■/■ = second experiment 




250 min. 



Fig. 1. Rate of disappearance of labelled phosphatide molecules 

fiom the plasma. 



in the blood, the number of labelled phosphatide molecules which an> 
decomposed in the plasma during the experiment can be neglected. 
The processes described above are going on under strictly physiological 
conditions. The replacement of ordinary phosphorus (^iP) by radioactive 
phosphorus (^^P) in some of the phosphatide molecules can certainly 
not be considered to entail the introduction of a non-physiological com- 
ponent into the circulation, as such a replacement cannot influence the 
chemical l)ehaviour of the phosphatide molecules to any significani 
extent. 

The rate at which the labelled phosphatides escape from the plasma 
of rabbits is seen in Tables 1 and 2, and also in Fig. 1. The figures of the 
tables were obtained by comparing the radioactivity of the phospha- 
tides present in 1 cc. plasma samples of rabbit B, taken at different 
intervals, with that of the phosphatides of an equal plasma volume 



264 ADVENTURES IN RADIOISOTOPE RESEARCH 

of rabbit A. The phosphatides were extracted by making use of Bloor's 
method. After being converted into phosphate by wet ashing, an aliquot 
part of the solution obtained was used in the colorimetric measurement 
of the P content, another to secure an ammonium magnesium phosphate 
precipitate, the activity of which was determined by a Geiger counter. 
The calculation of the amount of labelled phosphatides present in 
Ihe total plasma of the rabbit from that found in 1 cc. necessitates 
the knowledge of the total plasma volume. This was calculated from the 
blood volume and the known haematocrit value. The blood volume was 
determined by making use of a method recently described (Hahn and 
Hevesy, 1940). This method is based on the measurement of the dilution 
of a known volume of corpuscles containing labelled organic P compounds 
in the circulation of the animal, the blood volume of which is to l)c 
determined. In experiments on rabbits, the injection of foreign plasma 
was preceded by the removal of a corresponding volume of blood, as 
described above. In experiments on chicks, however, no blood was 
removed beforehand. 



EXPERIMENTS ON RABBITS(i^ 

(1) Some of the results obtained were previously published bj' us in a note to 
Nature 144, 204 (1939). — F. E. Haven and W. F. Bale [J. Biol. Chem. 129, 23 
(1939)] injected emulsions containing labelled phosphatides prepared from the 
liver of the rat into the circulation of another rat and found the labelled phospha- 
tides to accumulate mainly in the liver and the spleen. 

As seen in Tables 1 and 2 and also in Fig. 1 , half of the labelled phosphatides 
introduced into the plasma leave the circulation by penetrating through the capil- 
lary wall in the course of about an hour. As the non-labelled phosphatides can be 
expected to show the same behaviour as the labelled ones, we can conclude that, 
from aU phosphatide molecules present at the start of the experiment in the 
plasma, half will no longer be present after the lapse of about an hour, and will 
be replaced by others which were previously' located in the organs. 

Table 1. — Rate of Escape of Labelled 
Phosphatides through the Capillary 
Wall of a Rabbit Weighing 2.4 kgm 

First experiment 



Time 



Per cent of labelled phos- 
phatides injected into the 
jugular vein, present in 
the total plasma 



mill. 
30 „ 

82 ., 



100 

ti.5..S 
39.8 



120 „ 27.2 

247 „ . 17.6 



RATE OF PEXBTRATION OF PHOSPHATIDES THROUGH THE CAPILLARY WALL 2(55 

Table 2. — Rati; of Escape of Labellkd 

Phosphatides thkottgh the Capillary 

Wall of a Rabbit Weighing 2.8 kgm 

Serontl (experiment 



Timp 



Per cent of labelled phos- 
phatides injected into the 
jnc;ular vein, present in 
the total plasma 



niin 


1(»() 




85.fi 


llKi 


40.1 


242 


18.4 







Thico objections may be raised against the conclusions drawn above: a) Lal)!'!- 
led phosphatides can be decomposed in the plasma leading, for example, to the 
formation of labelled inorganic P; h) they can be incorporated into the corpuscles: 
c) they can be synthesised in the body of rabbit B, into which labelled plasma 
was injected. In that case, besides a loss of the labelled phos[)hatides introduced 
into the circulation of rabbit B, some gain of such phosphatitles due to a synthesis 
of labelled phosphatides in rabbit B would take place. 

The objections mentioned above are, however, not justified, as 

a) We recovered (see Table 6) more than 1/2 of the labelled phosphatides injected 
into the plasma of rabbit B 4 hours later in the organs investigated, in spite of 
the fact that the latter did not include the skin, the skeleton, and large parts 
of the digestive tract, which presumably took up an appreciable part of the labelled 
phosphatides. Furthermore, in the course of 4 hours, a non-negligible part of th(^ 
phosphatides present in some of the organs and, thus, also that of the labelled 
phosphatides taken up by these organs, was renewed. In the liver, about 
1/6 of the phosphatides piesent was found to be renewed in the course of 4 hours'. 
In view of the above considerations, the amount of phosphatides decomposed 
in the plasma in the course of a few minutes can certainly be disregarded. 

h) That in the course of a few houis the replacement of corpuscle phosphatides 
Vjy plasma phosphatides is a restricted one, is seen from the following figuies. 
In two experiments, after the lapse of 4 hours, 2 resp. 1.3 per cent of the labelled 
phosphatides originally present in the plasma of rabbits weie found to be located 
in the corpuscles. 

As to objection c), the formation of labelled phosphatides does not take place 
in rabVjit B to an\- significant extent in view of the absence of a sufficient amount 
of labelled phosphate. This fact is seen from the following consideration: We admi- 
nistered to rabbit A 5x10^ counts as phosphate and found the next day in the 
plasma of this rabbit 40,000 counts. We inje(!ted into rabbit B 20 cc. plasma 
containing 8000 counts, of which 4000 were due to phosphatide P and 4000 
to inorganic P. As from ox 10^ inorganic P counts introduced 20,000 phosphatide 
counts were found after the lapse of a day in rabbit A, we (^an conclude that, 
within that time, less than 20 phosphatide^ counts wci(> foinicfi in rabbit B, thus 
an insignificant amount. 



iQ. llEVE.SY and L. IIahn, Del Kgl. Dan-she Vidensk. Sel-skab, Biol. Mcdd. 
15, 5 (in40). 



260 



ADVENTURES IN RADIOISOTOPE RESEARCH 



EXPERIMENTS ON CHICKS 

Labelled phosphate was administered hy subcutaneous injection to chicks 
(Aj, Ag and A3, respectively). After the lapse of a day, plasma samples of these 
chicks were taken. One part (1 cc.) of the sample was injected into the jugularis of 
the chicks B^, C^, D^, E^, B^, C^, Dg a^^d B3, C3, Dg, Eg, Fg, respectively; another 
part was analysed. After the lapse of 7 to 67 minutes, plasma samples of chicks 
B, C, D, E and F, respectively-, were taken and the activity of their phosphatide 
content determined; heparin was added to the blood before it was centrifuged. 
In Tables 3, 4 and 5, the results of these experiments are recorded. The time recor- 
ded in Tables 3 and 5 was reckoned from the middle of the time of injection, 
which took about one minute. 



Table 3. — Pkrcentage of Labelled Phosphatides Present in the Plasma of 
Chicks Bj, C^, D^, after Injection of 1 cc. Plasma of Chick A^ Containing 

Labelled Phosphatides 



Chick 


'Weight of 
the chick 


Total plasma volume 


Time 


Per cent of the labelled 
Iihosphatide injected 




present in 
1 cc. pUisma 


present in the 
total plasmu 


B 


114 gm 
127 „ 
134 „ 


3.6 -f 1 cc. 
4.1 + 1 „ 
4.3 + 1 „ 


17.4 min 
17.9 „ 
17.0 „ 


8.02 
7.34 
7.12 


36 9 


2 
c, 


37 4 


^2 

D., 


37.7 



As seen in Table 3, after the lapse of 17.0 to 17.9 min, 1 cc. of the plasma of 
chicks Bj,, Cg and Dg, respectively, contains only about 7 per cent of the labelled 
phosphatide present in 1 cc. of the plasma of chick A injected into chicks Bg, C<, 
and Dg, respectively. This decrease is partly due to a dilution of the labelled phos- 
phatides present in 1 cc. by the non-labelled phosphatides present in about 4 cc. 
plasma of chicks Bg, C.2 and D^, and partly to an escape of the labelled phosphatides 
through the capillary wall into the organs and its replacement by non-labelled 
ones previously present in the organs. As seen in the last column of Table 3, from 
100 labelled phosphatide molecules introduced into the circulation of the chicks, 
only about 37 were present in the plasma after the lapse of about 17 min. 

Since the labelled phosphatides cannot be expected to show a different behaviour 
fiom the non-labelled ones, we can conclude that 63 per cent of all individual 
phosphatide molecules originally present are no longer in the plasma of the chick 
after the lapse of 17 min., being replaced by phosphatide molecules originally 
located outside the capillary wall. 

In the first experiment which we carried out on chicks (see Table 4), we ha\-e 
chosen another procedure. We compared the activity of 1 mgm phosphatide P 
extracted from 1 cc. plasma of chick A with the activity of 1 mgm phosphatide P 
extracted from 1 cc. plasma of chick B, C, D and E, respectively. Should the 
phosphatide concentration in the plasma of the different chicks used in this expe- 
liment be about the same, we could calculate from the data obtained the loss 
of labelled phosphatides through the capillary wall in the course of the first 7 
and the consecvitive 60 min. as well. When determining the phosphatide content 
of the plasma in our second experiment, we found, howcA-er, very pronounced 
differences between the plasma phosphatide contents of the chicks used. (Chick 



EATE OF PENETRATION OF PHOSPHATIDES THROIGH THE CAPILLARY WALL 267 

A2 = 6.5 mgm %; Bg = 7.5 mgm "0; ^o = 4.0 mgm %; D, = 4.8 mgin %)(i). 
From these variations in the phosj)hatide contents of the plasma we followed that 
from the data obtained in the first experiment, we cannot calculate the loss of 
labelled phosphatides by the plasma iu the course of the first 7 min., while we 
can state the loss sustained in the interval between 7 and 67 min after the start 
of the experiment. It is this vahu^ which is recorded in Table 4. 

T.\BL.E 4. — Change in the Specific Activity 
OF the Plasma Phosphatide of Chicks B^, Cj, Dj, Ej, 

AFTER THE INJECTION OF PlASM.\ OF ChICK A^ 

Containing Labelled Phosphatides 



Chink 


Weifjlit of chick 


Itiitio o£ specilic activitv 
of tlie pliospliatide P ob- 
tained after 7 and 67 miu 


B 


13S gm 
156 „ 
138(1) „ 
107(1) ^, 






2.2 


"1 

c 


2.1 


^1 

T), 


1.9 


1 
E 


2.8 


1 





*»> These chicks have shown pronounced exudates due to E-avitaminosis and were kiiully \mt at our 
disposal by Dr. H. Dam. The injection was kindly carried out by Mrs. Svendsen. The above I'isures do not 
permit us to draw any conclusion as to a difference in the permeability of, for example, the muscle capil- 
laries of normal chicks and chicks suffering from E-avitaminosis. To arrive at such a conclusion it wotdd 
be necessary to compare the labelled phosphatide content of the muscle tissue of normal clucks and of 
chicks sufferintr from E-avitaminosis at the end of the experiment. 



EFFECT OF HISTAMIN 

We also carried out experiments in which histamin was injected simul- 
taneously with the plasma containing labelled phosphatides. The results 
of" these experiments are seen in Table 5. 

The administration of histamin did not much affect the appearance 
of chicks C3 and D3, while chicks E3 and F3 could not stand on their 
feet for the first 5—10 min. which elapsed after the injection of histamin. 
From the last mentioned two chicks, only small blood samples, about 
0.4 cc, could be secured, while we collected several cc. from chicks 
which got no or only minor doses of histamin administered. The total 
plasma volume of the chick was calculated as described on p. 6. The 
average figure obtained for the labelled phosphatide content of one cc. 
plasma of chicks C3, D3, E3, and F3, to which histamin was administered, 
20 min. after the start of the experiment is aboul 7. It is the same 
figure which was obtaines for the labelled phosphatide content of the 
plasma of chicks B3 and Bg, Cg, Dg (see Table 3) in one cc. No striking effect 



(1) Comp. also the great variations in the phosphatide content of the blood 
of chicks found by F. \V. Lokexz, J. L. Ch.a.ikoff and C. Entenmax, J . Biol. 
Chcm. 123, 577 (1938). 



268 



ADVENTURES IN RADIOISOTOPE RESEARCH 



of the administration of histamin on the permeability of the capillaries 
by phosphatides is, thus, found. In view of the large fluctuations shown 
by the values obtained in the experiments in which histamin was ad- 
ministered, the above result is, however, to be interpreted cautiously. 

Table 5. — Percentage of Labelled Phosphatides Present in the Plasma 
OF Chicks B3, C3, D3, E^, and F3 after the In.iection of 1 cc. Plasma of Chick 

A3 Containing Labelled Phosphatides 





Weight oi the 
fliick 


Time 


mKin histamiii- 

dihydrochloride per 

gm chit'k weight 


I'ur cent i-it labelled phos- 
phatides injected, 
present in 


Chick 


1 PC plasma 


total plasma 

per gm 
body weight 


^3^^) 


113 gm 


21 mill. 





S.l 


37.8 


f'a 

R3 

E3 

F3 


104 gm. 
95 „ 
113 „ 
124 „ 


23 mill. 

23 „ 
9 

19 „ 


3 X 10 3 
5 X 10-3 
5 X 10^3 
1 X 10"^2 


5.5 

10.0 
4.0 

7 


23.8 
40.5 
18.5 
34.8 



<>' Conip. also Table :i. 



The permeability of different artificial membranes to phosphatides 
was investigated by Stjllmann and Verzar (1934). They found that 
through such membranes which are permeable to water blue and Congo 
red the plasma phosphatides can penetrate as well. 



UPTAKE OF LABELLED PHOSPHATIDES BY THE ORGANS 



In Ihe preceding chapters, we discussed the rate at which labelled 
phosphatide molecules located in the plasma penetrate through the 
capillary wall. We will now describe experiments which were carried 
out in order to determine to what extent the various organs took up the 
labelled phosphatides which left the circulation. We arrive at these 
figures by extracting the phosphatides of the organs and by determin- 
ing their activity. 

In Table 6, the percentage of labelled phosphatides introduced into the 
circulation, present at the end of the experiment in several organs, is 
recorded in the third column. The fourth column of the table contains 
data on the labelled phosphatide content of the interspaces computed 
on the assumption that all the labelled phosphatides present are to be 
found in the extracellular volume. For the extracellular volume of the 
organs of the rabbit we utilised the figures arrived at by Manery and 
Hastings (1939). In the fifth column, the distribution of the labelled 



RATE OF PENETRATION OF PHOSPHATIDES THROtOH THE fAl'IM.AHV \\.\U. 269 

phosphatides between equal volumes of the plasma and the extracellular 
fluid of the organs in question is stated on the assumption that the 
labelled phosphatides are solely to be found in the interspaces of the 
organ in question. The conclusion to be drawn from the figures of this 
column are discussed on page 270. 



Tabi.k tt. — Labkm.ki) Phosphatides Found in the Organs f>K Kabbit B aftkk 

THK Lapse of 4 Hours 



Organ 



Weight 



rercentago of tlie lal)ellfU 

phosphatides injected into 

the vein, present in 



the blood- 
tree organ 



1 (■<!. oxtra- 

oeUular 

fluid' 



Distribution eoel- 
ficient' of labelled 
phosphatides between 
equal volumes of 
extracellular water 
and plasma water 



Liver 

Kidneys 

Muscles 

Heart 

Spleen 

Small intestine mucosa 

Lungs 

Hrain 

Plasma 



02 


gm 


it 


99 


91(t 


99 


5 


99 


1. 


2„ 


4() 


., 


10 


95 


6 


J» 


79 


gm 



28.9 
0.88 
2.5 
0.21 
0.06 
1.1 
1.0 
0.05 



2.17 

0.19 

0.018 

(1.12 

O.K) 

0.065 

0.22 

0.022 



9.8 

0.85 

0.082 

0.54 

0.72 

0.29 

1.0 

0.10 



0.22 



• Calculated on the assumption that no penetration of labelled phosphatides into tlie cells took place 



In another experiment, only the labelled phosphatide content of 
liver and muscles were determined. The liver, weighing 85 gm, contained 
38 per cent of the labelled phosphatides injected after the lapse of 4 
hours, while in the blood-free muscles, weighing 1060 gm, 2.7 per cent 
of the labelled phosphatides administered were present. 

The figures given above relate to the labelled phosphatide content 
of organs of rabbits killed by bleeding. While such organs have only 
a comparatively small blood content, this cannot be entirely disregarded. 
Some of the labelled phosphatides present in the organs will be due to 
their blood content. In the muscles of the rabbit we found, by making 
use of the method of Eichelberger and Hastings (1937), that the 
blood content amounted to 0.5 per cent of the organs' weight. In the 
experiment described above, in which the weight of the muscles was 
1060 gm and the total plasma of the rabbit amounted to 97 cc, the 
blood present in the muscles contained 1.0 per cent of the labelled phos- 
phatides injected. In the case of the liver, the corresponding figure 
works out to be less than 1 per cent; in the case of the other organs 
the correction is insignificant. 



270 ADVENTURES IN RADIOISOTOPE RESEARCH 

As seen in Table 6, the labelled phosphatide content of all the organs 
but that of the liver can be interpreted as being present in the inter- 
spaces though this must not actually be the case. The liver contained, 
after the lapse of 4 hours, about ten times more phosphatides as can 
be explained by an uptake of the liver interspaces. This result suggests 
the explanation that not only the capillary wall but also the membrane 
of the liver cells is very easily permeable to phosphatides. The capillary 
wall of the small intestine, brain, and muscles is but fairly permeable, 
its permeability decreasing in the above sequence. The uptake of 
labelled phosphatides by 1 gm muscle makes out only about 1/170 
part of the labelled phosphatides taken up by 1 gm liver. The cor- 
responding figure for the small intestine mucosa is about 1/20. 

FORMATION AND EXCHANGE OF PHOSPHATIDES IN THE LIVER 

It is of interest to compare the amount of phosphatides synthesized 
in the liver with the amount which reaches the liver through an exchange 
process from the plasma. In the first case, we investigate the formation 
of labelled phosphatide molecules, in the second case no new labelled 
molecules were formed but all the labelled phosphatide molecules pre- 
sent were taken up by the liver from the plasma. This uptake is presum- 
ably followed by the release of a similar amount of phosphatide mole- 
cules previously present in the liver. An alternative explanation would 
be that the uptake of phosphatide molecules from the plasma by the 
liver is followed by a destruction of these molecules in the liver, the 
phosphatides lost by the plasma being replaced by phosphatides synthe- 
sised in other organs and liberated into the circulation. 

As found by us, in the course of 4 hours 150 mgm liver phosphatides 
were newly formed, while during the same time 52 mgm phosphatides 
are carried from the plasma into the liver; if this amount is not replaced, 
at least to a large extent, by an equal amount of phosphatides migrating 
in the opposite direction, then it must be supplied by another source 
to the plasma. The organ responsible for such a supply must be one in 
which phosphatide molecules are formed at an appreciable rate. This 
is primarily the case— besides the liver— in the small intestine. We have, 
therefore, to ask it the amount of phosphatides supplied during 4 hours 
by the intestine into the circulation suffices to compensate the uptake 
of phosphatide molecules by the liver from the plasma. Sullmanx 
and WiLBRANDT (1934) determined the amount of phosphatides carried 
into the circulation by the intestinal lymph of the rabbit. They found 
that up to 1/2 mgm phosphatide P can be carried by the lymph stream 
in the course of 4 hours, thus appreciably less than given off by the 
plasma to the liver during the same time. As the amount of phospha- 



RATE OF PEXKIHATIO.N Ul PHOSPHATIDES TJlKOKiH TJIE ( APILLAUV WALL 271 

tides brought inio the circulation from tlie intestine does not suffice to 
compensate the loss of phosphatides by the plasma due to the uptake 
of phosphatide molecules by the liver, we can hardly expect the amount 
released by other organs to compensate the loss of the phosphatides. 
We have thus, to conclude that in the liver not only a very marked 
turnover of phosphatides takes place, but that phosphatide molecules 
exchange also with great ease between the liver cells and the plasma. 



CALCULATION OF THE AMOUNT OF PHOSPHATIDES GIVEN OFF BY 

THE PLASMA TO THE LIVER 

We saw that, after the lapse of 4 hours, 29 to 38 per cent of the label- 
led phosphatide molecules originally present in the plasma were found 
in the liver of rabbits. We wish to calculate from the average of these 
figures the total amount of phosphatides which, originating from the 
plasma, reached the liver in the course of 4 hours. When calculating this 
amount, we must envisage that large amounts of labelled phosphatides 
were taken up by the liver and, to some extent, by other organs as 
well and were replaced by non-labelled ones. These process clearly lead 
to an increase of the sensitivity of the radioactive indicator in the course 
of the experiment. While, at the start of the experiment, 1 count indi- 
cates, for example, 1 fx mgm phosphatide P, at the end of the experiment 
it will indicate the presence of 5 ^ mgm. 

Let us denote by L^ the concentration of the labelled phosphatide 
molecules of the plasma at the start of the experiment, and by L^ that 
found after the lapse of t hours. The amount of phosphorus corresponding 
to Lq (average of the values obtained in two experiments) w-as found to 
be 2.4 mgm. The decrease of the labelled phosphatide content of the plasma 
is assumed to take place according to the equation 

where X is the constant of disappearance (analogous to the decay con- 
stant of radioactive bodies). If the liver alone would take up phospha- 
tide molecules from the plasma, the amount of labelled phosphatides 
which, coming from the plasma, were located in the liver, would be 
equal to X^ — L^. As this is not the case, w^e must determine experi- 
mentally the percentage of the labelled plasma phosphatides present 
in the liver at the end of the experiment, which we denote by E. To 
arrive at the figure giving the percentage of the total amount of 
plasma phosphatide molecules {X) which were found in the liver after 
the lapse of/ hours, we must multiply E by 



272 ADVENTURES IN RADIOISOTOPE RESEARCH 



1 — e-^' 
From X — 0.69 hour"', and / — 4 hours, it follows that 

y = 3. 

The value obtained for Y is too high, as the decrease of the labelled 
phosphatide content of the plasma takes place in the later stages of the 
experiment at a slower rate than according to the equation mentioned 
above. By taking into account this deviation we arrive at the values 

Y = 2.6 and X ^ 81 . 

From the fact that the phosphatide content of the plasma of the rabbit 
amounted to 60 mgm it follows that, from the phosphatide molecules 
present in the liver after the lapse of 4 hours, 52 mgm were such as migra- 
ted from the plasma into the liver during the experiment. 



Summary 

Plasma of rabbits containing labelled phosphatides was injected to other 
rabbits. Plasma samples of the last mentioned rabbits were taken at intervals 
and their labelled phosphatide content determined. The labelled phosphatide 
content of the organs was determined as well. The labelled phosphatides were 
found to disappear at a fairly rapid rate from the circulation. Half of those origi- 
nally present left the circulation in the course of about 2 hours. 

The labelled phosphatide molecules penetrate at a fast rate into the interspaces 
of the liver, at a much slower rate into that of other organs: the sequence of the 
decreasing rate of penetration being lungs, kidneys, spleen, heart, small intestine, 
brain, and muscles. 

The accumulation of labelled phosphatides in the Uver in the course of 4 hours 
was ten times larger- than expected in the case that the interspaces alone contained 
these phosphatides. From this fact follows a very great permeability of the cell 
walls of the liver to phosphatides. This is not the case for the other organs investi- 
gated. In view of the small amounts of phosphatides which penetrate, in the 
course of 4 hours, from the plasma into the muscles and the brain, we can conclude 
that the exchange of phosphatides between the cells of these orgarrs and the 
circulation is almost negligible. 

The total amount of phosphatides taken up from the plasma by the liver in 
the course of 4 hours was found to be 52 mgm. This uptake is accompanied by a 
migration of a similar amount in the opposite direction. Not only is the rate of 
turnover of phosphatides in the liver very high, the exchange of phosphatide mole- 
cules betweeir the liver cells and the plasma takes place at a much higher rate 
than the corresponding process between other organs and the circulation. 

In the course of 17 min, about 65 per cent of the labelled phosphatides origin- 
ally present in the plasma of the chicks left the circulation. 

Administration of large doses of histamin had no striking effect on the 
rate of penetration of phosphatides through the capillaries of the chick. 



Originally published in Kgl. Danske Videnskabenies Sel-skub- Biologinke Meddel- 

elser. 14, 2 (1938) 



31. ORIGIN OF PHOSPHORUS COMPOUNDS IN HENS' EGGS 

G. Hevesy and L. Hahn 

From tlio Institute of Theoretical Physics, University of (Copenhagen 

In this paper we discuss the origin of eggs phospliorus by making use 
of labelled (radioactive) phosphate as indicator. As the presence of 
labelled phosphorus in organic compounds proves that these compounds 
were synthetised since the administration of the labelled inorganic 
phosphate we can draw conclusions as to the place and time of for- 
mation of the lecithin and other compounds containing phosphorus 
and present in the egg, by making use of the above mentioned metluxf 
In the hope of finding which phosphorus compounds of the blood ar(> 
responsible for the formation of the lecithin and possibly other phospho- 
rus compounds of the egg we administered labelled sodium phosphate 
to hens by subcutaneous injection and investigated after some time the 
yolks removed from the ovary and further the composition of blood 
and of some of the organs. In other experiments eggs, laid at 
different times, were investigated. Finally we carried out also a few 
experiments in vitro. 

Several of the compounds building up the egg contain phosphorus. 
Lecithin and other phosphatides form about one tenth of the yolk of 
the hens egg, the ratio of P to that of the other elements present in these 
compounds being about 1 : 25. From the phosphoprotein of the yolk 
vitellin is the most abundant, it contains on the average 0.54% P. 
The total of phosphoprotein P present in the yolk is somewhat less 
than half of the phosphatide P present, while only small amounts of 
nucleoprotein P are found, as seen in Table l<i). 

The phosphatide P content of the average yolk amounts to 60 mgm 
and its total P content to about 94 mgm. The total P content of tlu^ 
yolk is thus about 0.6% of its total fresh weight, while that of the white 
of the egg is much smaller, amounting to about 0.01%. The P content 
per gm of the small yolks found in the ovary is appreciably lower as 
seen from Table 2 and increases with the increasing size of the yolk. 

(1' R. II. A. I'LiMMEii and F. M. ScoiT, Physiol. 38, 247. (1909.) 

IS Hevesv 



274 



ADVENTURES IN RADIOIRESEARCH 



The phosphorus content of the sheU oi hens eggs is very variable, 
fluctuating between 0.1 and 0.3% of the shell weight. It may be of 
interest to recall that on the average 50% of the weight of the hens 
eggs is due to albumin, 39% to yolk and 11% to the shell. 



Table 1. — Pho-sphorus Pre- 
sent IN THE Yolk in Percent of 
THE Total Phosphori:s 

I 

Phosphatide P i (il.4 

Water sohible P 9..1 



Phosphoprotein P 
Nucleoprotein P . . 



■Zt.ii 
1.6 



Table 2. — Phosphorus Content of Yolks 



Weight of 


Lec'itliin 1' in 


Total 1' ill 


LeiMtliin P in 


Total 


P in 


yolk in a.\n 


yolk in mgm 


yolk in mgm 


1 gm yolk 


1 gm 


yolk 


0.03 


0.03 


0.049 


1.00 mgm 


1.63 


mgm 


0.1 


0.20 


0.26 


2.00 „ 


2.67 




0.694 


1.72 


3..57 


2.49 „ 


5.18 




2..51 


6.25 


13.0 


2.49 „ 


5.2 




4.63 




4.00 




8.7 




7.68 




93.7.5 




12.2 




13.6 




125.0 




9.2 





^2' Lecithin jilus other phosphatides. 

According to general experience the yolk is formed while the growing 
egg is located in the ovary, about half of the white of the egg is formed 
by the albumin secreting portion of the oviduct, the shell membrane is 
deposited directly on this; and the more fluid portion of the albumin, 
constituting the second half of its entire l)ulk, enters through the shell 
membrane while the egg is in the isthmus and uterus. It has been found 
that the egg spends three hours in the glandular portion of the oviduct, 
one hour in the isthmus, sixteen to seventeen hours in the uterus includ- 
ing the time of laying. 

PHOSPHORUS COMPOUNDS IN HENS BLOOD 

The concentration of inorganic phosphorus, acid soluble phosphorus, 
lipoid (phosphatide) phosphorus and also of the total phosphorus pre- 
sent in blood, plasma and cells of chickens determined by Heller, 
Paltl and Thompson^^^ is shown in Fig. 1. The curves seen in the figure 



(i^V. C!. Heller H. 
357, (1934.) 



Paul, and R. B. Thompsen, T. Biol. Ghem. 106, 



OllIGIN or riiOs^i'HUitl S CUMl'Or.ND.s I.N HENS' EGGS 



275 




Acid-solubie Phosphorus in Plasma 

? — -<r — f 



60 90 120 150 180 210 310 34u 370 

Age of the chicken m days 



Fig. J. Pliospliorus distribution in chicken blood. 

were oljlained by analysing the blood of a large number of white Leghorn 
ehickens. The analyses were repeated once a month or oftener beginning 
at the time when the chickens were 1 month old and continuing through 
1he periods of growth, egg production, and subsequent molting. The 
results present very instructing data, they show that the phosphatide 
phosphorus alone, especially that of the plasma, changes very markedly 
with the age of the chicken, a rapid rise in the latter taking place after 
the lapse of 5 months at the time of produ(^tion, this high level being 
held under the entire production period with some fluctuations and 
dropping quickly as production ceases and molting season approaches. 



18* 



276 



ADVENTURES IN RADIOISOTOPE RESEARCH 



We determined the blood phosphorus of the laying hen denoted as I , 
the result being seen from Table 3. The blood phosphorus of another 
lien is discussed on p. 274. 



Table 3. 



P-CONTENT OF HeNS BlOOD 



iu plasma 



mgm "/(, 



mgin % 



ill oorpuscles iu blood 



Phospliatide P 

Inorganic P 

Total acid soluble P 
Rest (Protein) P . . . 




20.7 

21.3 

16.8 



In the blood of non-laying hens(J> after 24 hours fasting an average 
phosphatide P content of 16.8 mgm% was found the total plasma P 
amounting to 13 mgm%, the plasma inorganic P to 4.6 mgm%. 



EXPERIMENTAL METHODS 

The yolk wus dried by adding ice cold acetoii, the dry yolk was carefully 
pulverised and the powder obtained shaken for 15 min with 150 ce. ether, the 
last mentioned procedure being repeated four times using fresh ether. The ether 
was than carefully evaporated, the residue taken up with dry ether, the latter 
removed by evaporation, this time in a Kjeldahl flask, and the residue ashed. 

The phosphatides of plasma, corpuscles and total blood were extracted b_\- an 
ether-alcohol mixture after Bloor. The extract was several times carefully evapo- 
lated to dryness and taken up with ether or petrol ether. The residue of the first 
extraction was treated with trichloracetic acid (10 cc. of 10% solution for each 
cc. of blood) and from the filtrate obtained the inorganic P precipitated as ammo- 
nium magnesium phosphate; the esters present in the filtrate were hydrolised 
and the phosphate produced by the hydrolysis of the esters precipitated as ammo- 
nium magnesium phosphate. Though the extraction and the neutrahsation of the 
acid solution were both carried out at — 9°, some of the inorganic phosphorus 
present may be due to decomposition of the esters and we therefore gave in the 
table only the total acid soluble phosphorus present in the corpuscles which 
includes both the inorganic and the ester phosphorus. 

The liver was minced, dried in vacuo at room temperature, pulverised, 
dried again in vacuo and extracted with ether-alcohol (1:3), the latter being 
left to boil for 15 sec. In one case we extracted with ether alone to com- 
pare the active P content of the ether soluble phosphatides such as lecithin 
with that of the total phosphatides. The atad soluble P was extracted from 
the dried liver powder by treatment with cold ( — 10° to — 15°) solution of 
trichloracetic acid, first with a IO'^/q solution for 10 min and than twice 
w ith a 5 ^/q solution each for 5 min. The inorganic and organic constituents 
of the acid soluble phosphorus were separated as stated above. The P con- 
tent and activity of the residue obtained after extraction of the phosphatides 
and the acid soluble P was also investigated. 



1 H. M. DvKK and I. H. Hoe, J. Niitrit. 7, (523 (1<)34). 



ORIGIN OF PHOSPHORTS COMPOUNDS IX HENS' EGGS 277 

We detormined tho phosphoi'us contont of a known fraction of tlio inorganic 
phosphate soUilion ohiainod in Iho aV)ovo <lr>s(!ribo(l proeoduros V)y tho colorimol ric 
motliod of FisKE and Subakow. The pliospliato content o\' another fraction of the 
phosphate solution was precipitated in the form of ammonium magnesium phos- 
phate and its activity determined by making use of a Geiger tube counter. L(>t 
us say wo have administered a hen a hibelled phospliate sohit ion containing 1 mgm 
P and showing an acti\'ity of lO^ counts per niinut(\ Wo want to know what per- 
cent age of this labelled phosphorus will be found in tho yolk lecithin. To arrive 
at this figure we take from our solution containing the labelled phosphorus as 
much as corresponds to Vioooo ^^ ^^^ amount administered to the hen and preci- 
pitate the phosphate, denoting the piecijjitate obtained as our standard preparation, 
while wo will call tho precipitate obtained from the yolk lecnthin as leciithin prepa- 
ration. Before precipitating both the standard and the lecithin preparation we 
add to llie solution a known amount, usually about 80 mgm, of ina(!tive sodium 
phosphate, by so doing we diminish the amount of labelled phosphate possibly 
remaining in solution after precipitating with tho magnesium citrate reagents 
and furthermore we obtain a standard and a lecithin preparation of equal weight. 
The ^-rays emitted by the active phosphorus being to an equal extent absorbed 
in the two preparations the activity of which is to be compared, their weight 
and thus the thickness of the layers investigated being the same, there is no need 
to pay attention to the absoibtion of the /3-rays in the samples investigated. Nor 
need the decay of the radioactive P be considered, as both the prepaiations to be 
compared, the lecithin and the standard preparation, decay at the same rate. 
The yolk residue obtained after removal of the lecithin was treated in similar 
way and also the white of the egg, while the shell was ignited and dissolved after 
ignition in hydrochloric acid, the solution being tieated in the way described above. 
Tho samples were placed in small aluminium dishes having a surface area of 1.1 cm^ 
and were placed inunediately below tho aluminium window of the Geiger-counter 
used. 

Befoie discussing the results obtained we recall some facts about the circulation 
of labelled phosphorus in the blood. 

Sensitivity of labelling 

Let us start from labelled sodium phosphate preparation of such activity that 
when the later was fii"st put into the blood, I mgm P will show 10000 activity 
units. As a result of a rapid exchange going on chiefly between bone phosphate 
and the inorganic phosphate of the blood 1 mgm will soon correspond to loss 
than 10000 activity units. The total inorganics phosphate content of the blood 
remains (constant, except in the case which wo will not consider at present where 
a comparatively large amount is injected, while the individual phosphate ions 
will very soon be replaced to a large extent by other phosphate ions which were 
hitherto located in the skeleton or in other organs. After some time we shall find 
a large part of the labelled phosphate in the organs and the probaliility that the 
labelled phosphate leaves tho organs and gets back again into the blood will 
increase, the effect of this re-entrance into the blood will be that with increasing 
time the net rate of decrease of the inorganic labelled phosphate content of the 
blood will be less and loss. Loss of phosphate by excretion and by the formation 
of organic phosphorus compounds in the V)lood and in the oigaiis will fuithcr 
complicate the curve representing tho labelled P contont of the blood as a function 
of time. We determined tho latt(>r experimentally for the blood of different ani- 
mals and also of human subjects, but not for tho hen, (Compare, however, the 
results given on page 281). The conclusions drawn in this paper do not necessitate 



278 ADVEJfTURES IX EADIOISOTOPP; RESEARCH 



the knowledge of the change of the labelled phosphate content of the hens blood 
with time, it is for our present purpose sufficient to bear in mind that an initial 
rapid decrease of the labelled inorganic P content of the plasma occur and be- 
comes slower. 

In the first experiments described in this paper, in contrast to most of our 
experiments, we administered large amounts of P, of the order of magnitude 
of 100 mgm. The very strongly active phosphorus preparation (of a strength of 
about 10" counts) used in these experiments was a generous gift of Professor 
Lawrence and was prepared by the bombardment of few grams of red phosphorus 
by liigh speed eleuterium ions generated in Professor Lawrence's powerful cyclo- 
tron. The active P was thus mixed with a comparatively large amount of inactive 
phosphorus. In the experiments to be described, in contrast to some other experi- 
ments, the comparatively large amounts of phosphorus did not interfere, their 
presence in the active preparation has even the advantage that we can fix exactly- 
the limit within which the sensitivity of our indicator, the number of mgm of 
total inorganic P indicated by 1 count activity, varied throughovit the experiment. 
The 100 mgm P administered had an activity of 10** counts. If the labelled P had 
not been diluted bj- non-labelled P of the organs we should have found after the 
lapse of 28 hours, the time of the experiment discussed on page 276, a specific acti- 
vity of the plasma blood inorg. P— activity per mgm P— amounting to about 
1% of the total activitj^ administered. (The amount of inorg. P present in the 
total plasma is only about 5 mgm and thus much smaller than the 100 mgm P 
administered.) As seen from Table 9, however, only 0.01% was founel, showing 
that from the inorganic P atoms present in the blood of the hen aftei- the lapse 
of 28 hours only 1% were those actually administered, the rest being ones originat- 
ing from different organs and partly also from the food taken within that time. 

We carried out three types of experiments: 

a) Administration of labelled sodium phosphate to a hen and investigation 
of the eggs layed at different dates. 

h) Administration of labelled sodium phosphate, killing the animal, removal 
of the yolks and investigation of these yolks, the blood, the liver and other organs. 

c) Experiments in vitro in which eggs weie placed in labelled sodium phosphate 
solutions for few days and investigated as to what extent the labelled P penetra- 
ted into the egg. 

We will first discuss experiments of the type a). 

a) Investigation of the labelled phosphorus content of eggs laid at 
different dates 

We injected radioactive phosphorus as sodium phosphate subcutaneousl\- to 
hens and investigated the radioactive phosphonis content of the different parts 
of the eggs laid at different times. The first egg was layed 41/2 hours after admi- 
nistering the radioactive (labelled) phosphorus. We found the albumin to contain 
0.0015% of the 40 mgm of phosphorus injected, a similar amount 0.0014»o being 
present in the ^'olk. As the total phosphorus content of the yolk was found to be 
100 mgm and that of the albumin only 4 mgm, the specific activity (active phos- 
phorus per mgm normal phosphorus) was twenty-five times larger on the albumin 
than in the yolk. We found the lecithin phosphorus to be 53% of that of the total 
phosphorus of the yolk and to be entirely inactive. No synthesis of lecithin mole- 
cules took place in the yolk therefore within the 41/0 hours preceding the laying 
of the egg, as in that case some active lecithin molecules should have been formed: 
taking this fact into account the specific activity of the other than lecithin phos- 
phorus present in the yolk works out to be thirteen times smaller than that of 



ouToiN ov iMiospnoius roMrofXDS i\ hkns- e(;gs 270 

the albumin P. As 40 mgm active phosphorus wore injected and only O.OOOU mgm 
are found in the albumen we can (ioncdude that the formation of albumen from 
inorganic blood phosphorus in the course of the last A^i, hours which the egg 
spent in the oviduct is a very moderate one, even when we take into account 
that the O.OOOfi mgm active phosphorus found in the yolk passed through the 
albumen into the yolk bringing the amount of labelled phosphorus present at 
least temporarily in the albumen to 0.U012 mgm and that a large part of the active 
phosphorus injected gets rapidly replaced in the blood by non-active phos])horus 
present in the skeleton and other oigans. 

In the shell of the egg we find 10 mgm phosphorus b.\- chemical analysis (colori- 
metric method of Fiske and Subbarow) and 0.1 mgm of the labelled phosphorus 
administered by radioactive determination (measurements with a Geiger-counter). 
1 % of the shell phosphoms originates thus from the labelled phosphorus adminis- 
tered, which got into the shell in the ciourse of the last 414 hours before laying 
the egg. 

The labelled phosphorus content of eggs layed at different time is shown by 
the figures of the tables 4 to 6. 

In what follows we discuss the significance of these figures. That the specific 
activity of the shell is very much higher after 0.17 days than at a latter date 
is due to the fact that shortly after the administration of the labelled P the acti- 
vity of the inorganic P of the plasma is very high and it is the latter which is 
incorporated into the shell. As found by us in numerous cases the active P content 
of the plasma decreases first rapidly and later at a decreasing rate the differenc;e 
between the specific activity of the plasma and that of the tissues becoming less 
and loss. The specific activity of the shell phosphoi-us is a measure of that of the 
inorganic plasma P at the time of formation of the shell and vice v-ersa. The low 
specific activity of the albumin P in the egg layed after 0.17 days comes possibly 
for the following reasons. The white of the egg was already to an appreciable 
extent formed before the administration of the labelled P. The phosphorus compound 
of the plasma, presumably the plasma protein which mainly enters into the white 
was at such an early date after the administration of the labelled P active onh' 
to a small extent. The synthesis of labelled organic compounds takes some time 
and therefore shortly after the administration of labelled P the' speoificj activity 
of the inorganic plasma P is much higher than that of the organic P. On the other 
hand the labelled organic P flisappeare at a slower, usually even much slower, 
rate from the plasma than the labelled inorganic P, the latter having a unique 
opportvmity to exchange with the inactive tissue, especially bone tissue P. 

When comparing the yolk figures with those of the albumin we have to bear in 
mind that contrary to the albumin whic^h is formed within the day preceding the 
la^'ing of the egg the greater part of the yolk was already present when the active 
phosphorus was injected and therefore the labelled phosphorus of the yolk was 
fliluted by the unlabelled phosphoi-us already present in the yolk. With increasing 
time we should expect the amount of active phosphorus in the yolk to increase. 

Labelled P administrated at different dates 

In another set of experiments we were interested in producing strongly active 
egg-lecithin to find out whether after feeding the latter iis dry yolk to rats, the 
presence of active lecithin in the blood of the rats can be ascertained. This was 
found not to be the case. In these experiments we administered to the hen on 
several days active phosphorus which made the interpretation of the activit_\- 
measurement of the yolk removed from the ovary rather difficult. A compaiison 
of the activity of the shell of the yolk with its fluid interior revealcti large diffe- 



280 



ADVENTURES IN RADIOISOTOPE RESEARCH 



rences. The semi-solid yolk shell formed from very active blood was found in 
one case to be seven times more active than the fluid interior of the yolk, and 
five times in another case. With decreasing size of the yolk the difference between 
the specific activity of the yolk phosphorus originating from the inner and llio 
outer part of the yolk diminished and finally vanished. 

Table 4. — Active Phosphorxts Content of Eggs. Hen I. 



Percentage of active phosphorus administered found in ; 



Egg laid after ad- 
ministration of active 
pliosphorus 



Shell 



AUiumin 



Total yolk Yolk lecithin 



0.17tlay.s 
1.0 „ 
3.0 ,. 
4.5 „ 
6.5 ., 



0.24 

0.052 

0.036 

0.026 

0.022 



0.0015 

0.032 

0.030 

0.027 

0.020 



0.0014 
0.100 
0.42 
0.95 

0.85 



0.000 

0.014 

0.17 

0.34 

0.35 



Table 5. 



Hen I. 



Percentage active phosphorus administered found in 1 mgm egg phosphorus x 10^. 

(Specific activity x 10=). 



Egg laid after administra- 
tion of active phosphorus 


Shell 


Albumin 


Yolk after 
removal of 
phosphatides 


Yolk 
phosphatides 


0.17day.s 


24.0 
5.2 
3.6 
2.6 
2.2 


0.38 

8.0 

7.5 

6.8 

5.0 


0.03 

2.0 

5.1 

12.6 

10.4 





1.0 „ 


0.26 


3.0 „ 


3.3 


4.5 


<i.4 


6.5 ., 


7.0 







Table 6. — Distribution of the Active Phosphorus Administered 
between Different Parts of the Egg 



Egg laid after administra- 
tion of active phosphorus 



;<hell 



Albumin 



Yolk after 

extraction of 

phosphatides % 



Yolk lecithin 
phosphatides % 



0.17 days 

1.0 „ 

3.0 „ 

4.5 „ 

6.5 „ 

0.5 (lay.s 

3 
4 
6 



98.9 

27.0 

7.0 

2.6 

9 r^ 



46.1 

30.5 

15.0 

7.2 



Hen I. 

0.6 
17.0 
6.0 
2.7 
2.2 

Hen II. 

35.0 

6.8 
6.2 

2.8 



0.5 
48.8 
50.9 
60.5 
56.0 



0.0 

7.2 

36.1 

34.2 

39.3 





18 


.9 


38.2 




24.5 


56.4 




32.4 


58.5 




31.5 



OKIGIN OK PHOSPHORUS COMPOTXDS IN IIENS' EGOS 



281 



b) Specific activity of yolk phosphorus 

We administered to a hen 100 ingin. I' iis sodium-phosphate showing an activity 
of 10^ counts and killed the hen after the lapse of 28 hours. From the ovary 34 
yolks were removed and from the oviduct one egg. The weights of these are recor- 
ded in Table 7. 



Table 7 







Weight 


of 


yolk 


Specimeps 
present 


About 


•( 

' 30 

100 

700 

2500 

5000 

7500 

13000 

18000 


mgm . 

99 

99 






20 








9 




























; ■ 
















(Kgg) 


J? 















The specimens of 700 mgm and more weie treated separately while averages 
of the 30 mgm and the 100 mgm. yolks were taken. The lecithin was extracted 
by ether and the residue brought into solution as described above. The results 
obtained are seen in Table 8. 

The specific activity of the total P shows a maximum in the case of the 2500 
mgm yolk. This result, puzzling at first sight, can be easily understood after consi- 



Table 9. ^ Specific Activity of Yolk Phosphorus 
(Percentage of the activity administered present in 1 mgm P) 



Weight of yolk 



Phosphatide P 

% 


Xon Phospliati- 
de P 

o/ 
/o 


Total P 


0.00055 


0.018 


0.0073 


0.00814 


0.0173 


0.0129 


0.0147 


0.018(i 


0.01(54 


— 


— 


0.0090 


— 


— 


0.0055 


— 


■ — 


(1.0044 



30—100 mgm 
700 

2500 

4(500 

7700 
13(500 



dering Fig. 2 taken from a paper of H. GerhabtzI, in which the daily increase 
in weight of the yolks of a hen is recorded. The yolk grown from active blood 
and thus active will be diluted by the non-active yolk already present and this 
dilution will load to a decrease of the specific activity of its P content. The dilution 
being least in the case of the 2500 mgm yolk, (comp. Fig. 2) its specific activity 
is bound to be highest. It takes some time after administration of the labelled 
sodium phosphate until labelled lecithin is transported into the plasma whereas 



' H. Gerhartz, Arch, dtsch. Ges. Physiol. 156, 215 (1914). 



282 



ADVENTURES IX RADIOISOTOPE RESEARCH 



inorganic P of very high activity is piesent ahnost at once after injecting the 
active sodium phosphate. The non phosphatide P of the yolk is partly inor- 
ganic P which gets into the yolk in the early stage of the experiment when its 
specific activity is very high; we mvist therefore consider the lecithin P and not 
the non- lecithin or total P content as a proper measure of the growth of the 
A oik. From the fact that the lecithin P of the 30—100 mgm yolks became active 

only to a very slight degree we 
must conclude for example that 
they had hardly grown within tlK> 
last 28 hours. When discussing 
the labelled let-ithin P (phospha- 
tide P) present in blood and in 
some of the organs we shall find 
definite evidence that the yolk 
lecithin is dra^^^l from the plasma 
lecithin. 

It is of interest to remark that 
the ratio of the total active leci- 
thin content of small yolks, such 
as would require 10 daj's or more 
to attain completion, is a quanti- 
tative measure of their relative 
growth since the administration 
of the labelled P. When, however, 
comparing the lecithin P activity 
of a small yolk which increases 
its weight in the course of a da^- 
only to a slight extent with that 
of a large yolk growing at a rate of 
few gm per day the ratio of the 
total activities will not always be 
a correct measuie of the growth 
since the administration of the la- 
belled P. It may happen (comp. 
Fig. 2) that the giowth of yolk per 
hour is larger at the end than in 
the beginning of the experiment the latter process determining thus to a larger 
extent the total growth within the time of experiment. From which it follows, 
that if at the beginning of the experiment the specific activity of blood lecithin 
happens to V)e greater than at the end, we underestimate tlie growth of the 
large yoUc. 

It is, however, the determination of the slow rate of giowth of the small and 
tiny yolks, often present in a very large number in the ovary, which can be of 
special interest and whidi can hardly be determined by any method other than 
that outlined above. 




Fig. 2. Increment of the weight 

of yolks in the coui-se of 1 2 da_\'S 

before completion of the yolk 

according to Gerhartz. 



Investigation of blood phosphorus 



Plasma and (corpuscles of the hens blood were separately investigated using 
the experimental method described on page 276. The results obtained are seen from 
Table 9 which contains data on the specific activity (activity per mgm P in percent 
of that injected) and also the total phosphorus present in the hens blood under 



ORIGIN OF J'HOSPItOIUS COMPOUNDS IX HENS' EfiGS 283 

the assumption (hat tho vohunr of blood of the hon iiniounl(>(l to 150 cc. and tho 
volume of tho hlood plasma to 100 eo. 

Table 9. — Spkcific Activity' and I'otal Phosphori's Contkxt 

OF THK IIkn's Blood 



I Specific 
Fraction 



I 



activity 



Total pliosphorus 
content 



Plasma inorganif (1.0104 ! 5.4 

Plasma phosphatide 0.012r, j 20.0 

Corpuscles phosphatide 0.004f) 1 1.3 

Corpuscles acid .soluble i 0.003<'> ^O.r, 

Corpuscles protein 0.0031 j I.k'J 



That the specific activity of the plasma phosphatide P is greater after 28 hoiiis 
than that of the inorganic P is due, as discussed on page 279, to the rapid disappoa- 
i-ance of the individual inorganic P atoms from the plasma. In the experiment 
discussed on page 286, in which the hen was killed only 5 hours after the admi- 
nistration of the labelled P, the specific activity of the phosphatide P was found 
to be only 42% of that of the inorganic P. 

It is of great interest that the specific activity of the plasma phosphatide P 
is several times larger than that of the corpuscle phosphatide which shows that 
a much smaller percentage of the corpuscle phosphatide than of the plasma phos- 
phatide is renewed in the course of the experiment. This is an interesting result 
as it definitely disposes of the often discussed possibihty that the blood phospha- 
tide is synthetised in the corpuscles. Some of the corpuscles being formed during 
the experiment from labelled plasma are bound to contain labelled phosphatides; 
labelled phosphatides can furthermore easily get into the stroma of the corpuscles 
which are partly composed of phosphatides. 

A very suggesting change in the phosphatide content of hens blood at the 
time of production was ascertained by Heller, Paul, and Thompson (comp. 
Fig. 1). The most interesting feature of the curves recorded by them is a gradual 
increase in the total P of the blood at the time of production, this high level being 
held during the entire production period with some fluctuations and dropping 
quickly as production ceases and molting season approaches. The increase is duo 
to that of the lipoid P and is much more conspicious in the case of the plasma 
than in that of the corpuscles; the lipoid P content of tho plasma is higher all 
through than that of the corpuscles, at the peak of production tho foi-mer value 
being nearly throe times higher than the last mentioned one. As about 2/. ^ of tho 
blood volume is composed of plasma it follows, that from the total lipoid P present 
in the blood ^/g arc to bo found in tho plasma. The predominance of phosphatide 
i' ir the plasma found for laying hens is entirely unique as seen from the figures 
of Table 10, but understandable if we envisage the great strain put on the organism 
of a hen as to lecithin suppl;^'. A hon laying daily has to produce about (50 mgm 
lecithin! P a day: taking a total plasma volume amounting (o 100. cc the total 
lecithin P of the plasma works out to be 20 mgm. If (he loc^hhin found in the yolk 
is, as suggested from our tosuKs, taken from the plasma lecithin tlu n the plasma 
has to give off three times its total lecithin content in tho course of a day thus 



1 Lecithin plus other phosphatides. 



284 



ADVE.VTURES IN RADIOISOTOPE RESEARCH 



putting an appreciable strain on the lecithin circulation. A strain which would 
be still more pronounced in the case of a lower plasma lecithin content. 

Table 10. — Phosphatide P in Plasma and Cells or 
Different Animals 



mgm % P in 



I'lasmii 



Cell 



Ratio 



cell 



plasma 



Rat 

Ral)bit 

Man 

Dog 

Laying hen 



2.6 
3.3 

9 
14 
14—20 



10 
12 
19 
14 
8—23 



3.S 
3.(i 
2.1 
I 

0.87 



Protein phosphorus in the hen^s blood 

After removal of the phosphatides and the aciid soluble phosphorus, the remain- 
ing P is generally assumed to be present as protein P. The protein P content 
31.8 mgm% found in the corpuscles of the hen in the 28 hours experiment is much 
higher than in the corpuscles of the blood of other animals, the corpuscles of the 
rabbit containing for example, as found by Mr. Aten, 4.4 mgm %. The same 
considerations apply to the protein P content of the plasma, which was found 
to amount to 9.4 mgm% for the blood of the hen in question and of 7 mgm % in 
the case of the hen discussed on page 276 while the plasma of a rabbit, for example, 
was found to contain only 0.03 mgm % piotein P. From the high value of the 
specific acti\'ity of the protein P in the 5 houis experiment it follows that the 
protein phosphorus compounds present in the plasma were renewed even at a 
higher rate than those of the phosphatides. This result suggests a great participa- 
tion of the plasma phosphorus protein in the formation of the egg. To arrive at a 
final conclusion as to the relation between the phosphorus protein compounds 
of the plasma and those of the yolk and white is difficult because of the fact 
that we lack simple methods of separation of the protein compounds. Vitellin, 
for example, can only be isolated by a very tedious and lengthy process and the 
isolation and separation of the blood protein phosphorus compounds are still 
more difficult, partly because onl^• small quantities of these substances can be 
secured in the ex})eriment. The fact that we have to base our conclusions on 
the amount of phosphorus present in the residue, remaining after extraction 
of the phosphatides and the acid soluble phosphorus compounds makes the result 
obtained less trustworthy than those arrived at when investigating the phospha- 
tides, for example. The high value for the protein phosphorus of the corpuscles 
found by us, which may to some extent be due to an incomplete separation ot the 
phosphatides and acid soluble P, is supported by the data obtained by Heller, 
Paul and Thompson. They find for the total P present in the cells of laying 
hens about 100 mgm %, but only about 40 mgm % for the sum of inorganic acid 
soluble and lecithin P. The discrepancy suggests the presence of a further not 
investigated P fraction, which might be protein P. In the case of the plasma 
phosphorus the curves of Heller and his colleagues show the anomaly mentionetl 
only to a smaller degree; the total phosphorus found by them is not very much 
larger than the sum of the acid soluble and phosphatide P. 



ORIGIN OF PHOSPHORUS COMPOUNDS IN HENS' EGGS 



285 



The high protein P content of the blood plasma of a lading hen has presumably 
the same Viiologieal significaneo as the high phosphatide P content, namely a 
reduction of the strain put on the protein resp. phosphat ide prochicing and eari_\ ing 
system in the organism of laying hens. 

Phosphatide content of the liver 

We extracted the total phosphatide content of the livei- of a hen 28 hovirs 
alter t ho administration of the labelliMl P, using the method described on page 277. 
Since we were interested in seeing whether lecithin soluV)]e in ether shows the 
same specific activity as the total phos{)ha1 ides we exlraded another part of 
the liver tissue with ether alone. W'e found no marked difi'erence, as s(>en from 
Tables 11 and 11a, which also ccntain data on the specific activity of inorganic 
and acid soluble (other than inorganic) V of the liver. 

As seen from Table 11 the specific; ativity of the liver phosphatide P is 5(5% 
of that of the inorganic P, from which it follows that about one half of the phos- 
phatide molecules are labelled and thus formed after administral ion of the lab(>ll(>(i 

Table 11. — vSpkcific Activity of thk Livkr P 
(Activity per mgni. P) 



Kiai-tioii 



Specific uclivitj 



Total phfjsphatides (ether-alcohol extract) 

Lecithin (ether extract ) 

Inorganic 

Acid soluble, other than inorganic 



0.0152 
0.015S 
0.0272 
0.0224 



sodium phosphate. This result must, however, be interpreted with great caution. 
As already mentioned on page 277 in the early stage of the experiment the specific 
activity of P of the plasma is much higher than in the latter stages and the inorga- 
nic P of the liver was also more active at the early stage. This change of the speci- 
fic; activity with time would not affect our lesults if the specific activity of the 
phosphatide P shovild decrease with time at the same rate as does that of the 
inorganic P. That is, however, not the case. The phosphatide molecmles can mainly 
• scape from the circulation at an appreciable rate into the yolk, while the indivi- 
(kial inorganic P atoms present in the plasma can rapidly exchange with such 
piesent, for example, in the skeleton, the latter being a much faster proc^ess in 
view of the huge extent of the skeleton. Therefore, when drawinir conclusion 



Table ila — Percentagk of Labelled P Administkreu 
Found in Plasma, CoRPrscLEs and Liver 



Fraction 


Total Plasma 
(100 gm) % 


Total Corpus- 
cles (50 gm) % 


Total Liver 
(44 gm) % 


Phospliatide P 


0.2.-) 


0.0.52 


(I.Od.S 


Inorganic P 


0. ().-)(•) 


— 




Total acid solnljle P 


— 


0.100 


l.c,4:? 


Protein P 


0.1 7r> 

0.4S2 


0.0.5(1 
(».202 




Total P 


2.251 







J LI 



yu. 



286 ADVENTURES IN RADIOISOTOPE RESEARCH 

from the comparison of the specific activities of the phosphatide P and the 
inorganic P as to what extent the phospliatide molecules got labelled we are 
apt to get values which are possibly too high. A trustworthy value could be 
obtained by keeping the specific activity of the inorganic P of the plasma 
constant by continuous injection of labelled phosphate of varying concentration 
and by thus avoiding a decrease in the specific activity of the inorganic P 
of the plasma, which is used for the synthesis of the phosphatide molecules 
in the liver and elsewhere. In the above case we can, however, conclude that 
a very appreciable part of the liver phosphatide molecules most have been re- 
newed within the 28 hours of the experiment. In experiments on isolated livers 
in which the skeleton and other organs are not present it is easy to calculate 
from the ratio of the specific activities of inorganic P and phosphatide P the 
amount of newly formed phosphatides. In an isolated liver of a cat in the 
course of 2.5 hours about 1% of the phosphatide molec-ules present are newly 
formed. If in the course of 2.5 hours in an isolated liver of a cat about 1% 
of the phosphatide content is renewed there can be hardly any doubt that in 
the liver of a living hen in the course of 28 hours a large part of the phos- 
phatide found is synthetised during that interval: in the liver of a living animal 
the enzymatic and other actions necessary for the synthesis of phosphatides 
are certainly as abundant as in an isolated liver and the phosphatide formation 
in the liver of a laying hen could hardly be less than in that of a cat. We are led 
to the same conclusion by the following consideration. The daily amount of phos- 
phatide P tranfered from the plasma into the ovary is, in the case of the hen in 
qviestion, which was lading one egg every other day, about 50 mgm. The main 
source of phosphatide production is, as we wiU see, the Uver, and an amount 
not very far from 60 mgm must therefore have been produced daily in the hver 
of the hen. Since the latter containing 38 mgm of phosphatide P, a large amount 
of the liver phosphatide must have been renewed during the experiment. A similar 
conclusion applies to the plasma phosphatides, the 50 mgm phosphatide P being- 
carried by the plasma, the total content of which is 20 mgm, the plasma phospha- 
tide molecules must have been renewed repeatedly. 

We are thus led to the result that the main source of phosphatides in a laying- 
hen is the liver and that more than one half of the phosphatide molecules present 
in the hens liver were newly formed during the 28 hours preceding the admini- 
stration of labelled phosphate. That the greatest part of the phosphatides is formed 
in the liver of a laying hen and reach the ovary through the plasma is very clearly 
shown in an experiment in which the hen was killetl only 5 hours after admi- 
nistration of the labelled phosphate. 

The acid soluble P of the liver, other than inorganic, mainly derived from P 
ester, shows, as seen in Table 11, a higher specific activity than the phosphatide 
P present in the liver. 

Experiment on a hen killed after five hours 

3.8 cc. of physiological sodium chloride solution containing 10 mgm labelled 
sodium phosphate were injected subcutaneously into a hen which weighed 1800 
gms. The hen, which layed previously one egg daily weighing about 45 gm was 
kiUed after the lapse of 5 hours. The results obtained are seen in Table 12. Two 
separate determinations were carried out, the values found and also their 
average are given. 

As seen from Table 12 the specific acti\ity of the phosphatide P, which is a 
measure of newly formed phosphatides, is by far the greatest in the liver and 
markedly higher than that of the plasma phosphatide P. Contrary to the 28 hour 



OHKil.N OF l'll()SJ'JlOia>; CO.Ml'OIMJS J.\ JIK.NS' K(;(;S 



28: 



oxporiniont, wlunc^ the peiconta^o of 
pUisma nearly icac-hod tliat found i 
concentration gradient in the flow 
liver into the plasma is very clearly s 
led molecules in the ovary phos- 
phatide is, on the other hand, 
much smaller than in the j:)lasma 
phosphatides. From this it follows 
that the labelled phosphatide mo- 
lecules present in the ovary were 
within 5 hours only partly leplaced 
by ones present in the plasma. 
We investigated a yolk weighing 
1.0 gm. The figures obtained are 
given in Table 12. A second yolk 
investigated w'eighed 2.7 gm. and 
its lecithin P had a specific acti- 
vity of 0.0050. The specific act- 
vity of the yolk lecithin of the first 
mentioned yolk was found to be 
about Vii of that of the plasma 
lecithin. From these figures it 
follows that about Vii of the 1.0 
gm i. e., 0.09 gm of yolk were 
giown within 5 hours. The actual 
growth was, however, presumably 
greater than 0.09 gm, since in 
the early stages of the experiment 
the plasma phosphatide was only 
very shghtly active and so was the 
yolk tissue formed in this phase 
of its development. The fact that 



newly formed phosphat i(l(> molecules in the 
n the liver, in the 5 hours experiment the 
of labelled phosphatides directed from the 
liown (comp. Fig. 3). The percentage of label- 



spieen 




— PiQ' mo 



---^3^ Inrestine 



Fig. 3. The heaviness of the sha- 
ding indicates the specifier activity 
of the lecithin P and thus the per- 
centage of the phosphatide mole- 
cules formed within the last five 
hours in the total phosphatides of 
the organ in question. 



Tablp: 12. — Specific Activity of Phosphatide P 



Organ 


Specific activity ( % of aotivity 
given, found in 1 mgm P) 


Relative specific 
activity ; that of the 
inorganic plasma 1* 
taken = 1 




Single values 


Average 


Liver } 

Plasma < 

Ovary i 

Yolk 1 


0.094 ) 
0.082 i 
0.0(M> ) 
0.0(50 ) 
0.00()4 ) 
O.OOM i 
o.oor):{ j 
0.0075 \ 

O.OIS } 

0.018 j 

<-" 0.02 


0.08S 
O.OHO 
0.0064 
0.00t)4 

O.OIS 
<0.02 


0.54 
0.4:} 
0.031) 
0.025 


■ 


Intestine < 

Spleen 


0.11 
< 0.1 









288 ADVENTURES IN RADIOISOTOPE RESEARCH 

the specific at^tivity of the ovary phosphatide was found to be low, as low as 
that of the yolk, proves definitely that the role of the ovary is not produc- 
tion of phosphatides but their extraction from the blood plasma together 
with other suitable constituents. The combination of phosphatides with proteins 
giving the characteristic composition and consistency of the yolk, is 
evidently one of its principal functions. In the experiment described above 
the specific activity of the P of the yolk soluble in trichloracetic acid was 
found to be 0.035, thus 1/4 5 part of that of the inorganic P of the plasma 
the latter being 0.16. Making the assumption that most of the atad soluble P 
originates from the inorganic P of the plasma we find a growth of the yolk amount- 
ing to 1/45 part of its weight of 1.0 gm during the experiment. ^Miile the above 
mentioned figure of Yii was, as ahead}- mentioned, a lower limit of the part of 
the yolk newly formed within 5 hours, the figure of I/4.5 is a higher limit. A part 
of the acid soluble yolk phosphorus was formed at an earlier stage when the speci 
fie activity of the plasma inorganic P was appreciably higher than at the end 
of the experiment, and as our calculation is based on the specific activity of the 
plasma inorganic P at the end of the experiment it gives too high a value for the 
amount of yolk formed during the experiment. 

The phosphorus of the white of the egg removed from the oviduct had a low 
specific activity, namely 0.0013. This is an interesting result in view of the strong 
activity shown by the phosphorus compovinds present in the plasma (comp. Table 
13). A possible explanation of this result is that some of the phosphorus present 
in the protein or other compounds of the oviduct tissue is utihsed to produce the 
phosphorus compounds present in the white of the egg. In the course of five houi's 
perhaps the compounds present in the tissue of the oviduct get labelled only to 
a slight extent. An other explanation is that while the average plasma protein 
P has a high specific activity 0.15 after the lapse of five hours, the specific activity 
of the phosphorus of one of the components of the protein mixture might be low. 
We are now engaged in the investigation of the origin of Ihe phosphoius 
present in the white of the egg. 

Table 13. — Specific Activity of 
Plasma Phosphor rs 



Fraction Specific activity 



Inorganic P 
Lecithin P . 



(I. If) 

(I. ()()'.) 



Protein P <I.14 



c) Experiments //( vitro 

We placed eggs in a neutral physiological sodiumphosphate solution containing 
30 mgm P for 24 hours and investigated the activity of the different parts of the 
eggs, the results being seen in Tables 14 and 15. 

The comparatively high labelled P content of the shell is due to phosphate 
exchange processes between the large shell surface and the solution and possible 
also to the formation slight amounts of calcium phosphate trom the carbonate 
of the shell. An investigation of the activity of the lecithin extracted from the 
yolk gave an entirely negative result, this in agreement with the observation 
recorded on p. 279 that after the egg left the ovary no more lecithin formation 
takes place. 



ORIGIN OF PHOSPHORUS COMPOUNDS IN HENS' EGGS 



289 



Table 14. — Ratio of the Specific Activity of Egg P and 

SoLTTIOX P 





Shell Allnniiin 


Yolk 


Egg I (Total P) 

Egg II (Total P) 


l.S X 10-1 
2.0 X 10~i 


1.4 X 10~3 

1.5 X 10-3 


1.9 X 10-6 
4.0 X 10^6 


Table 15. — Distribution of the Active Phosphorus Taken 
UP between the Different Parts of the Ego 




Shell 1 Albumin 
% % 


Yolk 
% 


Egg I 

Egg II 


99.46 
99.40 


0.44 
0.41 


0.10 
0.19 



DISCUSSION 

We saw that by investigating the labelled phosphorus content of 
eggs or yolks we could draw conclusions as to the growth of the egg 
or yolk since the date of administration of the labelled P. It is, for 
example, possible to show that while the egg is in the oviduct not only 
is no more yolk formed but also no new lecithin molecules are synthetised. 
Should suitable enzymes be present, new and thus labelled lecithin 
molecules could be formed without any growth of the yolk. The ovary 
of a laying hen contains numerous tiny yolks growing at a slow rate; 
by comparing the incorporation of labelled P by such yolks, we get 
a quantitative measure of their relative growth since the administration 
of the labelled P. When comparing the growth of small yolks with 
large ones we can usually not obtain strictly quantitative results as 
to the relative growth because of the much more rapid relative growth 
of large yolks compared with that of small ones. 

Placing eggs in a solution containing labelled P for some days we 
find the shell to contain an appreciable part of labelled P, while the 
amount shown by the white and especially by the yolk is very small, 
though easily measureable, even in the case of the yolk. No formation 
of labelled lecithin is, however, found in the yolk. 

As to the formation of lecithin in the growing yolk, we arrive at the 
following result: The phosphatides found in the yolk are synthetised 
at least to a large extent in the liver and are transported through the 
plasma to the ovary which extracts the phosphatides. This is most 
clearly seen in the experiment in which the hen was killed only 5 hours 
after the administration of the labelled sodium phosphate. In this experi- 
ment the specific activity of the liver phosphatide P reached 54% of 



19 Hevesy 



290 ADVENTURES IN RADIOISOTOPE RESEARCH 

that of the plasma inorganic P, while the specific activity of the plasma 
pliosphatide P was appreciably smaller, amounting to only 43%; that 
of the ovary was very much smaller, namely 3.9%, and about as large 
as that of the strongest yolk phosphatide P. In the 28 hours experiment 
as to be expected, the difference in the specific activities was much 
smaller, the specific activity of the liver phosphatides being only some- 
w^hat higher than that of the plasma phosphatides. In the 28 hours 
experiment on the hen which used to lay one egg every other day the 
amount of phosphatides passing through the plasma on the way into 
the ovary was, in the course of the experiment, about twice the amount 
of phosphatides present in the plasma. In the 5 hours experiment, in 
which the hen experimented on was laying one egg daily, the amount, 
of phosphatide passing the plasma on the way into the ovary was about 
half the amount present in the plasma. From the low specific activity 
of the phosphatide P, that is from the low percentage of newly formed 
phosphatide in the ovary, it follows that in this organ only an insigni- 
ficant amount of phosphatide can be formed. We have also to consider 
that a part of the labelled phosphatides found in the ovary is due to the 
presence of blood containing the latter. The specific activity of the 
plasma phosphatide P being appreciably smaller than that of the liver 
the labelled phosphatides must have come from the liver into the blood 
and not vice versa. By carrying out experiments in vitro with blood 
containing labelled sodium phosphate we found only a slight formation 
of labelled phosphatides, which is in accordance with the above con- 
clusion. 

The formation of phosphatides in the intestinal mucose by using 
radioactive phosphorus as indicator was first shown l)y Aetom, Perrier, 
Santagello, Sarzana and Segre*^). They found in an experiment, 
carried out on a rat, that after injecting labelled sodium phosphate 
the phosphatides extracted from the gut after a few days showed a 
specific activity only exceeded by that of the liver phosphatide P, the 
ratio of the specific activities being 1.2. The phosphatide production in a 
laying hen is larger than in any other animal of similar size, as the amount 
produced daily to be incorporated in the yolk is as much as about 2 
times that present in the liver which contains more phosphatide than 
any other organ. The laying hen is, therefore, a very suitable animal 
for studying phosphatide formation. In our 5 hours experiment the 
specific activity of the intestinal phosphatide P is much smaller than 
that of the liver phosphatide P and also than the plasma phosphatide 
P. The bulk of the labelled P present in the plasma can, therefore, not 
originate from the intestinal phosphatide and the latter can not be the 
chief source of the yolk phosphatide. The phosphatides formed in the 

(i^iYaiure 139, 836(1937). 



ORIGIN OF I'HOSPHORT'S COMPOU^'DS IX HENS' EGGS 291 

« 

intestine can, liow(^ver, hav(> and presumably actually do have a role 
in the suppl}^ of .the plasma i)hosphatides.The presence of phosphatides 
in the intestinal lymph was repeatedly shown*') in experiments on dogs. 
The amount of phosphatides reaching the hens circulation by the influx 
of intestinal lymph could be ascertained by measuring the amount 
of intestinal lymph produced and also its phosphatide^ content. In Fig. 3 
we show the specific activities of the phosphatide P in the organs 
of the hen killed 5 hours after the administration of the labelled sodium 
phosphate. The heaviness of the shading indicates the specific activity. 
A hen laying daily deposits about 60 mgm phosphatide P in the yolk 
or about 3 times as much phosphatide as present in the plasma. In the 
course of a day the phosphatide content of the plasma of a laying hen 
must therefore be replenished three times. In view of this great strain 
on the phosphatide circulation in the plasma it is very significant that 
the plasma phosphatide content of a laying hen is higher than in most 
other animals. If the laying hens plasma should show such a low phos- 
phatide content as does a rabbit or a rat (per cc.)the plasma lecithin would 
have to be replenished as much as 17 — 22 times a day. It is significant 
that the high phosphatide content is maintained only during the laying 
period and that the red cells contain less phosphatide than the plasma, 
a behaviour not shown by the blood of any other animal investigated. 
We find furthermore that in the course of 28 hours taken by the experi- 
ment a much greater part of the phosphatides found in the plasma is 
labelled than of that contained in the corpuscles. This is a significant 
result as it demonstrates clearly that lecithin is carried to the ovary 
by the blood plasma and not the blood cells which obtain their lecithin 
in various ways. Labelled phosphatide could be taken up by the cell 
membrane, possibly diffuse through the cell membrane; labelled inorga- 
nic phosphorus which was found by us to diffuse at a moderate speed 
into the corpuscle could lead to the formation of labelled phosphatide 
phosphorus inside the latter, finally the lecithin could get into the cor- 
puscles at their birth. If they are formed from labelled plasma the newly 
formed corpuscles should become labelled as well. As to the formation 
of labelled phosphatide from labelled inorganic P in blood, we found, 
in experiments in vitro that such a formation actually takes place, 
though only on very minute scale. As to the rate of formation of blood 
corpuscles, some information on this point could be obtained by inject- 
ing labelled plasma and investigating the radioactivity of the phosphorus 
compounds isolated from the corpuscles after the lapse of some time. 
If after the lapse of a day, for example, only 1% of the corpuscle phos- 
phatides were found to be labelled we could conclude that the rate of 

(i) H. E. Hamerich, Amer. J. Physiol. 114, 342 (1934); S. Freeman and A. 
C. Joy, loc. cit 110, 132 (1935). 

19* 



292 ADVENTURES IN RADIOISOTOPE RESEARCH 

formation of the corpuscles per day is less than 1% of the total corpuscles 
present. 

As to the white of the egg, we find that at least a large part of its 
phosphorus content is drawn from organic phosphorus compounds, 
possibly from protein phosphorus. We arrived at this result by compar- 
ing the specific activity of the phosphorus of the white of the egg with 
that extracted from the shell. The latter derives its phosphate content 
from the inorganic P of the blood plasma and is accordingly a convenient 
measure of the activity of the latter. The shell is formed at about the 
same time as the white of the egg, the great discrepancy between the 
specific activity of the shell P and albumin P exclude the possibility 
that they are of common origin. 



Summary 

By administering labelled sodium phosphate to laying hens the share of the 
labelled phosphorus administered in the formation of the yolk, albumin and shell 
of the egg can be followed by aid of radioactive measurements. The comparison 
of the specific activity (activity per mgm P) of the phosphorus extracted from 
blood plasma phosphatides with that extracted from the hver, the ovary, and the 
\'olk phosphatides leads to the result that the buUc of the phosphatides of the 
yolk originate in the Hver. It gets from the liver into the plasma and is then taken 
by the latter to the ovary. 

No formation of phosphatides takes place in the oviduct. After the egg leaves 
the ovary no more active phosphatide is formed. No formation of labelled phos- 
phatide in the yolk can be ascertained in experiments in which an egg is placed 
for a day in a labelled sodium phosphate solution. In the last mentioned experi- 
ment in vitro shght amounts of labelled phosphorus are found in the yolk, appre- 
ciable quantities in the white, and large amounts in the shell. 

The specific activity of the phosphatides extracted from the blood corpuscles 
was found to be only 1/3 of that extracted from the plasma. Therefore, we conclude 
that the phosphatides formed in the hver and other organs are carried to the 
ovary by the plasma rather than by the corpuscles. The latter apparently play 
no important role in this process. 



Originally published in Biochcm. J. 32, 2147 (1938) 

32. THE ORIGIN OF THE PHOSPHORUS COMPOUNDS 
IN THE EMBRYO OF THE CHICKEN 

G. C. Hevesy, H. B. Levi and 0. H. Rebbe 

From the Institute of Theoretical Physics, University of Copenhagen 

Several of the numerous compounds containing phosphorus present 
HI the embryo of the chicken^ occur in the yolk and the white of the 
egg. Those which do are chiefly phosphatides and nucleoproteins but, 
as Table 1 shows, other phosphorus compounds also occur in those 
parts of the egg. 



Table 1. — [Plimmer -;^ Scott, 1909]. Percentage 

OF THE Total, P (94 mgm) at the Beginning and 

the End of Incubation of a Hen's Egg 



1 Beginning 


End 


Inorganic P 

Water-soluble P 

Ether-soluble P 

Vitellin-P 

Nucleoprotein-P 


Trace 

6.2 

64.8 

27.1 

1.9 


60 

8.6 
19. .3 


12 



KuGLEE [1936] has lately found that, on the twentieth day of incu- 
bation, i. e. the last day but one, only 25 mgm of the 65 mgm of lipoid 
P originally present in the yolk remained there; 8 mgm were found in Ihe 
embryo, and the remainder had been hydrolysed yielding inorganic P. 
About two-thirds of the phosphatides present were found to be lecilliin 
and one-third kephalin. In view of the large store of phosphatides present 
in the yolk even shortly before the egg is hatched, we should expect 
the embryo to avail itself of this store when it n(MHls phosphatides to 
build up its nervous system and other organs containing these substan- 
ces. We can test this point by introducing labellcMl (radioactive) sodium 
phosphate into the egg before incubation and investigating if and to 
what extent the phosphatide of th(> yolk and of the (Mubrvo become 



1 A detailed investigation of the soluble pho.sphorus compounds piesenl in Ihe- 
embryo of the chicken was lerenlly jiublislicd by Xeedham ct nl. (1937). 



294 ADVENTURES IN RADIOISOTOPE RESEARCH 

labelled. If the yolk piiosphatide remains unlabelled while that of 
embryo becomes radioactive, we can conclude that the phosphatide 
molecules present in the embryo have not come from the yolk but 
have been built up in the embryo with the participation of labelled 
inorganic P. Similar considerations apply to certain other compounds 
occurring in the embryo. 

METHODS 

The phosphorus content of a series of solutions is usually determined 
colorimetrically. For example, the inorganic P present in one sample 
of an acid-soluble fraction can be determined in this way, and then in 
another sample the phosphagen-P present can be converted into inor- 
ganic P, so that colorimetric determination now supplies the value for 
the inorganic P + phosphagen-P. In our experiments this was inade- 
quate. We had to measure not only the P content but also the activity 
of the various fractions, so we had to obtain precipitates in each case. 
To obtain sufficient precipitate when dealing with eggs only incubated 
for a few days, it was necessary to work with several eggs simultaneously. 

We precipitated the phosphorus, after bringing it into the inorganic 
state, as ammonium magnesium phosphate. The precipitate was then 
dissolved in 0.1 N HCl and an aliquot part was sucked into a glass 
cuvette. This was placed below the Geiger counter used to determine 
the activity of the preparations, while another aliquot part was utilized 
for the colorimetric determination of the phosphorus content. The glass 
cuvettes were covered with a thin mica window (5—6 mgm per cm'^) 
which only absorbed to a negligible extent the B-rays emitted by the 
radioactive phosphorus; the area of the mica window was I.l cm- and 
the liquid content of the cuvette amounted to about 0.5 ml. 

We were interested in the determination of the activity of 1 mgm P 
prepared from different phosphorus compounds present in the embryo 
or in the remains. Accordingly we were not concerned with quantitative 
determination of the P compounds present but concentrated our 
efforts on obtaining the various fractions in a pure state — to avoid, 
for example, traces of inorganic phosphate remaining in the phospha- 
tides extracted from the yolk. As the phosphatides of the yolk were 
found to be but slightly active, while the inorganic P was strongly active, 
even a small contamination of the former by the latter was to be avoided. 
The white, the yolk, the embryo and, in some cases, the amniotic and 
allantoic liquids were worked up simultaneously. 

As regards the white we were only interested in the total activity 
present after incubation. The white was ignited (reduced to ash) and its 
phosphorus precipitated as ammonium magnesium phosphate. 



OMGIX OF THE PHOSPHORUS COMPOUNDS IX EMBRYO OF CHICKEN 295 

The yolk was dried with acetone and the phosphatides extracted three 
times from the dry product with a 3:1 alcohol-ether mixture. The 
alcohol and other were then evaporated off at about 50° hi vacuo and 
the residue was taken up with light petroleum and lil1(>ro(l. The filtrate 
was evaporated //? vacuo, the residue ignited, and the phosphorus 
precipitated as ammonium magnesium phosphate. 

Another part of the yolk was treated as follows. The acid-soluble 
compounds were extracted, then the phosphatides were removed as 
described above, and the residual part containing mainly vitelUn-P 
and nucleoprotein-P was ignited; the P content of this last part was 
determined as ammonium magnesium phosphate. 

The embryos were dropped, immediately after being removed from 
their eggs, into liquid air and were subsequently pulverized. The embryo 
powder was then extracted several times with cold trichloracetic acid— 
in the first two extractions a 10% solution was used, and later one oif 
5%. The extract was filtered into cold concentrated NaOH solution 
and divided into three parts, (a), (6) and (c). From {a) a sample of the 
average acid-soluble P of the embryo was secured, {b) was precipitated 
with 25% barium acetate solution at pR 6.5. The cold precipitate was 
washed with a dilute barium acetate solution, centrifuged and dissolved 
in a few drops of cold HNO3. The inorganic P present was then precipi- 
tated by adding Fiske's reagent. The remaining filtrate was hydrolysed 
with N HCl at 100° for 7 min. to split the two labile phosphate radicals 
of adenosinetriphosphoric acid. The phosphorus set free was finally 
precipitated as ammonium magnesium phosphate, Barium hydroxide 
was added to the filtrate from the barium precipitation to remove any 
inorganic P, the precipitate was separated by centrifuging and ethyl 
alcohol was added to the remaining liquid until an alcohol concentration 
of nearly 60% was reached. The precipitate obtained after addition 
of alcohol [OsTERN et al., 1936] contained the hexosemonophosphate. 
Its P content was determined in the usual way. The third part, (c), 
was hydrolysed with N HCl in the presence of 0.1 M ammonium 
molybdate for 30 min. at 40°. In the course of 30 min. most of the 
phosphagen present decomposed, so that the inorganic P originally pres- 
ent as such, and that obtained by the decomposition of the phosphagen^, 
were secured together in this fraction. 

After removal of the acid-soluble P the embryo was thoroughly treated 
with an alcohol-ether mixture, as described above, to remove the phos- 
phatides. The residue, containing mainly nucleoprotein-P, was ignited 
with concentrated sulphuric and nitric acids and the P precipitated in 
the usual way. 

(i^On the phosphagen content of the embryo of the chicken, cf. Lehmann 
and Needham [1937]. 



296 



ADVENTURES IN RADIOISOTOPE RESEARCH 



RESULTS 

Eggs incubated for 6—18 days. The results of the determination of the 
specific activities (activities per mgm P) of the different fractions 
extracted from seven embryos and from the remaining parts of eggs 
incubated for 11 days are shown in Table 2, while Tables 3—5 give 
the results obtained with eggs incubated for 18, 16 and 6 days. In 
addition to the specific activity (activity per mgm P, with that of the P 
extracted from the white of the egg taken as 100), we have also recorded 
in Tables 2 and 3 the activity (in counts per minute or in % of amount 
injected) and the P content of the fraction — this last quantity being 
determined, in all cases, by the method of Fiske and Subbarrov^. 



Ta ble 2. — Specific Activity of P Extracted from Different Fractions of an 
Egg Incubated for 11 Days. (Specific Activity of P Extracted from the 

White Taken as 100) 



Fraction 



Counts 
per min 



Specific 
activity 



Embryo: Average acid-soluble P 

Inorganic P 

Adenosine-P -|- inorganic P . . 

Creatine-P 

Phosphatide-P 

Residual ("nucleoprotein") P 
Yolk: Phosphatide-P 



0.074 
0.077 
0.121 
0.171 
0.561 
1.49 
10.4 




Table 3. — Specific Activity of P Extracted from Different Fractions of an 
Egg Incttbated for 18 Days. (Specific Activity of P Extracted from the White 

Taken as 100) 



Fraction 



I 



mgm P 



% of amount 
injected 



Embryo: Average acid-soluble P 

Inorganic (without skeleton) P 

Tibia and femur-P 

Adenosine-P 

Phosphatlde-P 

Residual ("nucleoprotein") P . . 
Yolk: Acid-soluble P 

Phosphatide-P 

Residual P 



19.7 
10.91 
4.50 
0.048 
1.08 
0.204 
0.828 
17.50 
2.16 



53.5 
27.2 
7.6 
0.14 
1.7 
0.3 
1.3 
0.28 
0.12 



Specific 
activity 



19 

17 

11 

20 

11 

10 

11 
0.11 
0.40 



The figures for the specific activities (activities per mgm P) of different 
fractions extracted from an embryo and from the remaining parts of an 
egg incubated for 18 days are shown in Table 3. The P content in mgm, 



ORIGIX OF THE PHOSPHORI'S COMPOUNDS IN E.MBKYO OF CHICKEN 



297 



the percentage of the injected activity present in the fraction and the 
relative specific activity are recorded; the specific activity of the P 
extracted from the white of the egg is taken as 100. 

The specific activities obtained when the eggs were incubated for 
16 and 6 days respectively are seen in Tables 4 and 5. 

Table 4. — Specific Activity of P Extracted Fractions 

OF an Egg Incubated for 16 Days. (The Specific 
Activity op P Extracted from the White Taken as 100) 



Fraction 



Specific activity 



Embryo: Average acid-soluble P 

Inorganic (without skeleton) P 

Tibia and femur-P 

Creatine-P 

Hexosemonophosphate-P 

Phosphatide-P 

Residual („nucleoprotein") P . . 
Yolk: Acid-soluble P 

Phosphatide-P 

Residual P 



14 

14 

15 

14 

19 

12 

16 

12 
0.14 
1.22 



Table 5. — Specific Activity of P Extracted from 
Different Fractions of 10 Eggs Incubated for 6 
Days. (Specific Activity of Embryo Phosphatide P 

Taken as 100) 



Fraction 



Specific activity 



Yolk: 



Embryo: Phosphatide P 

Average (phosphatide) P 

Inorganic P 

Acid-soluble minus inorganic P 

Phosphatide P 

Residual P 



100 

113 

60 

34 

0.032 

1.3 



As the figures show, the phosphatides extracted from the yolk are 
only slightly active, while those extracted from the embryo show strong 
activity 1 mgm of embryo phosphatide-P is at least 100 times as active as 
1 mgm yolk phosphatide P. Furthermore, the specific activity of the 
embryo phosphatide-P is about as high as that of the embryo inorganic 
P, showing that an inorganic P atom reaching the embryo has about 
the same chance of entering the skeleton as of being incorporated in 
a phosphatide molecule by an enzymic process— which of the two 
systems it enters is governed solely by probability considerations. 
From this it follows that the phosphatide molecules in the embryo are 



298 ADVENTURES IN RADIOISOTOPE RESEARCH 

not identical with those derived from the yolk, but are synthesized 
in the embryo. 

The formation of labelled phosphatides in growing eggs was investigated 
by Hevesy and Hahn [1938]. It was found that the phosphatides 
present in the yolk are taken up from the plasma by the ovary and 
incorporated into the latter; as soon as the yolk leaves the ovary no 
more change occurs in the content or composition of its phosphatides. 
When labelled phosphate is administered to a hen after the yolk has 
left the ovary and is located in the oviduct, the egg takes up active 
phosphate but no active phosphatide is formed. In experiments in vitro 
as well, eggs placed in radioactive sodium phosphate solution take up 
active phosphate Vmt no active phosphalides are formed. The slight 
activity of the phosphatides present in the yolk of incubated eggs is 
presumably due to the influx into the yolk of small amounts of active 
phosphatides synthesized in the embryo. This view is supported by the 
fact that the ratio of the specific activities of the embryo phosphatide 
P and yolk phosphatide-P was much larger (3000) in the 6 days experi- 
ment than in the 16 clays experiment (100). The activity of the residual 
P of the yolk, which is mainly composed of vitellin and nucleoprotein, 
was larger than that of the phosphatides; this can be understood if we 
admit the possibility that the extraction of the strongly active, non- 
protein constituent of the yolk is not quantitative, for in this case the 
specific activity of the residual P would be increased. 

The embryonic residue obtained after extraction of the acid-soluble 
and ether-soluble constituents is composed chiefly of nucleoproteins. 
That the specific activity of the nucleoprotein-P is the same as that 
of the inorganic P extracted from the embryo is not surprising, because 
much less nucleoprotein is present in the yolk than in the embryo (Table 
1). The greater part of the nucleoproteins present in the embryo must 
therefore have been built up in the course of incubation; during this 
process labelled phosphate has an opportunity of entering the nucleo- 
protein molecules. 

Distribution of radioactive phosphate in the egg 

The greater part of the sodium phosphate injected into the white 
is still found at the end of the 6 days experiment in that part of the 
egg. The distribution of the activity between white, yolk, connecting 
fluids (which were not, however, free from wliite and yolk) and embryo 
is seen in Table 6. 

The low activity of the yolk might possibly be due to a slow rate of 
penetration of the vitellin membrane by the phosphate ions; this point is 
under investigation. Another possible explanation is that the inorganic 
P content of the yolk is lower than that of the white. If a distribution 



ORIGIN OF THE PHOSPHORUS COMPOUNDS IX EMBRVO OF CHICKEN 



299 



equilibrium is reached, the activity should be proportional to the amount 
of inorganic phosphate present in the phase in question, since the inor- 
ganic P, among all the P compounds present in the yolk and white, 
is practically the only source of activity; in the 6 days experiment, for 



Table 6. — Distribution of Injected 

Active Phosphate between 

Different Parts of the Egg 



Time of 
incubation 


Fraction 


% activity 


6 days 


White 


61.6 




Yolk 


10.3 




Liquids 


26.00 




Embryo 


1.7 


16 days 


White 


14.9 




Yolk 


1.7 




Liqtxids 


19.8 




Embryo 


63.0 



example, 10% of the 10.3% activity found in the yolk was present as 
inorganic P. Finally we have to envisage the possibility that a part of 
the inorganic phosphate injected is not freely movable in the white — it 
might be precipitated as calcium phosphate or attached to proteins, 
its mobility being lowered thereby. 

We have also carried out experiments in which 0.1 ml. physiological 
NaCl solution containing a negligible amount of labelled sodium 
phosphate was injected into eggs which were not incubated. After the 
lapse of 5 days the distribution of the activity in different parts of the 
egg was determined; 97% was found in the white and 3% in the yolk. 
As was of course to be expected, a still greater preference for the white 
was shown by the active phosphorus in this experiment; the duration 
of the experiment was shorter than that of those discussed above, and 
transport of phosphorus from the white to the embryo was 
absent. 

To test whether the water injected encountered any hindrance in its 
propagation through the egg, we injected 0.2 ml. heavy water into the 
white of the egg; after the lapse of 5 days water was distilled separa- 
tely from the white and from the yolk and the densities determined. 
We are much indebted to Mr O. Jacobson for carrying out the density 
determinations using Linderstrom— Lang's float method. He found 
that the water prepared from the white had a density exceeding that 
of normal water by 484 parts per million, while the corresponding figure 
for the water obtained from the yolk was 437. The deuterium content of 
the water distilled off from yolk was found to be only about 10% lower 



300 ADVENTURES IN RADIOISOTOPE RESEARCH 

than that of the water from the white, showing that in the course of 
5 days the water injected w^as very nearly evenly distributed throughout 
the egg, in contrast to the injected active phosphate. The anomalous 
behaviour of the latter, while of interest in the study of the circulation 
of phosphate ions in wdiite and yolk, in no way influences the investigat- 
ion of the main problem discussed in this paper— namely, if and to 
what extent the molecules of the different phosphorus compounds 
present in the embryo are built up there or are drawn, from the yolk. 



Introduction of labelled hexosemonophosphate into the egg to be incubated 

In one set of experiments, instead of following up the fate of labelled 
inorganic P in incubated eggs, we introduced radioactive hexosemono- 
phosphate. Prof. Parnas very kindly presented us with this compound 
(prepared by Dr Ostern) in the form of barium hexosemonophosphate, 
from which, by treatment with sodium sulphate in the cold, the sodium 
compoundof the ester was obtained. 0.2 ml., containing about 0.2 mgm 
P as hexosemonophosphate salt and about 3 mgm. sodium sulphate, was. 
injected into the white of each of the eggs to be incubated; to avoid 
decomposition of the ester, the solution was kept ice-cooled until it was 
injected into the egg. Of the 10 eggs receiving this treatment, only two 
supplied living embryos. After a lapse of 14 days, 7.7% of the activity 
injected was found to have been incorporated in the embryo (5.8% in 
the yolk) and a large fraction was also to be found in the white and in 
the connecting liquids. If, of the various fractions extracted from the 
embryo, we had only found activity in the fraction containing hexose- 
monophosphate, we should have had to conclude that the hexosemono- 
phosphate does not decompose in the egg but enters the embryo as 
such. In view of the results obtained in the experiments carried out 
with labelled inorganic phosphate, however, such behaviour was hardly 
to be expected. Furthermore, Kay [1926] found that in the embryo 
tlie phosphatase activity of the developing bone was extremely high, 
the phosphatase decomposing the hexosemonophosphate. We isolated 
the hexosemonophosphate from the embryo, as described on p. 295, 
and compared the specific activity of this fraction with that of the 
inorganic phosphate ( -}-creatine-P). We also isolated the phosphatide 
fraction and the residual phosphorus fraction containing mainly nucleo- 
protein-P. As Table 7 show^s, no conspicuous difference can be seen 
between the specific activities of the different fractions of the embryo, 
with the possible exception of the residual P. In these experiments small 
activities had to be measured and the differences found between the 
first three fractions lie within the errors of the experiment. The results 
obtained suggest the explanation that active inorganic P splits off 



ORIGIN OF THE PHOSPHORUS COMPOUNDS IN EMBRYO OF CHICKEN 301 

i'rom the labelled hcxomonophosphate injected and is incorporated in 
the different phosphorus compounds of the embryo, while the hcxomono- 
phosphate molecules extracted from the embryo are not those syn- 
thesized by Dr Ostern but are molecules built up by the chicken's 
embryo. 



Tablk 7. — Specific Activity of P from Different 

Fractions from Two Eggs Incubated for 14 Days after 

THE Injection of Radioactive HexosemonoPhosphate. 

(Specific Activity' of P Extracted from the White 

Taken as 100) 



Fraction 



Specific activity 



Embryo: Inorganic P 

Hexosemonophosphate-P . . . . 

Phosphatide-P 

Residual ("nucleoprotein") P 
Yolk: Inorganic P 

Hexosemonophosphate-P . . . . 

Phosphatide -{- residual P . . . 



24 
26 
20 
11 
36 
18 




The low value found for the residual P of the embryo may possibly 
1)0 due to the building up of a part of the nucleoprotein fraction at an 
early date before much of the active hexosemonophosphate introduced 
has decomposed. The phosphatide-P and residual P extracted from the 
yolk were found to be inactive. These fractions were found to be only 
slightly active even after the injection of strongly active inorganic P, 
and the absence of activity after the injection into the egg of the much 
weaker hexosemonophosphate was only to be expected. The hexose- 
monophosphate fraction isolated from the yolk had a specific activity 
of 18; the inorganic P, 36. The larger value found for the specific activity 
of the inorganic P is possibly to be explained in the following way. 
Some active hexosemonophosphate diffuses into the yolk and partly 
decomposes into active inorganic P: this is the source of most of the 
active inorganic P which we isolated from the yolk. The hexosemono- 
phosphate, isolated by the method outlined on p. 296, contains, besides 
the active hexosemonophosphate, some non-active hexosemonophos- 
phate and possibly also some other acid-soluble P compound separated 
simultaneously, which diminished the specific activity of the "hexose 
monophosphate" fraction isolated from the yolk. In the embryo, on 
account of the strong enzymic action prevailing there, all phosphorus- 
compounds become labelled; on the other hand, in the yolk, as we 
have just mentioned, no such labelling takes place. 



302 ADVENTURES IN RADIOISOTOPE RESEARCH 

On the phosphatide synthesis in the embryo of the chicken 

We saw that the phosphatide molecules present in the chicken's 
embryo are not identical with those formerly located in the yolk, 
but that they were synthesized in the embryo. The work of Schonheimer 
and RiTTENBERG [1936] gives us important information about the units 
which are utilized in the synthesis. They found, by making use of deute- 
rium as an indicator, that the developing hen's egg forms no new fatty 
acids and their result excluded also the possibility that unsaturated 
fatty acids present in the egg had been hydrogenated during develop- 
ment. Needham [1931], on the other hand, found that a marked desatu- 
ration occurs in an aqueous emulsion of embryonic tissues mixed with 
the corresponding yolk and vigorously shaken. The embryo must thus 
make use of the fatty acids present in the yolk to build up its phosphati- 
des: in doing this it possibly gives some preference to the less saturated 
fatty acids. The fatty acid components of the phosphatides extracted 
from the embryo are found to be less saturated than those extracted 
from the yolk residue. This, at first sight puzzling fact that the embryo, 
instead of using the phosphatide molecules found in great abundance 
in the yolk synthesizes its own phosphatide molecules, becomes less 
puzzling when we envisage the likely possibility that the synthesis of 
phosphatide molecules is a step in other chemical processes which 
occur simultaneously in the growing embryo. 

Summary 

Radioactive sodium phosphate was injected into lien's eggs which were then 
incubated in some experiments for 6, and in others for 11, 16 and 18 days. While 
the phosphatide -phosphorus extracted from the embryo always showed a high 
specific activity (activity per mgm P), that extracted from the yolk was hardly 
active at all. The phosphatide molecules present in the embryo could not there- 
fore have been taken from the yoUi only, but must have been sjTithesized in the 
embryo. The investigation of the "acid-soluble" and residual (mainly nucleoprotein) 
phosphorus extracted from the embryo led to a similar result — namely, that 
the ratio in which the labelled inorganic phosphorus atoms are incorporated 
into the different phosphorus compounds present in the embryo is governed 
solely by probability considerations. Practically all the phosphorus atoms present 
in the various compounds of the embryo must pass through the stage of inorganic 
P; only the inorganic phosphorus present in the embryo is taken as such from 
the yolk or the white. 

In some experiments, instead of radioactive sodium phosphate, labelled hexo- 
semonphosphate was injected into the egg before incubation. The hexosemono- 
phosphate-phosphorus extracted from the embryo had about the same specific 
activity as the inorganic and the phosphatide phosporus extracted. This result 
suggests that inorganic phosphate radicals which were split off from the hexo- 
semonphosphate and from other compounds present in the yolk and the white, 
rather than the hexosomonophosphate molecules introduced into the latter, are 
utilized to build up the phosphorus compounds of the chicken's embryo. 



ORIGIN OF THE PHOSPHORUS COMPOUNDS IN EMBRYO OF CHICKEN 30;i 



References 

Hevesy and Hahn (1938) Kgl. danfike vidensk. Seh-kah. Biol. Medd. 14, 1. 
Kay (in2()) Brit. J. exp. Path. 7, 177. 
KuGLER (1936) Amer. J. Physiol. 115, 287. 
Lehmann and Needham (1937) J. exp. Biol. 14, 483. 

Needham (1931) Chemical Embryology, Vol. II, p. 1171. Univoiity Press, Cam- 
bridge. 
Needham, Nowinski, Dixon and Cook (1937) Biochem. J. 31, 11. 
OsTERN, GuTHKE and Terszakowec (1936) Hoppe-Seyl. Z. 243, 9. 
Plimmer and Scott (1909) J. Physiol. 38, 247. 
ScHONHEiMER and RiTTENBERG (1936) J. Biol. Chem. 114, 381. 



Originally published in Nature 142, III (1938) 



33. FORMATION OF MILK 

A. H. W. Aten and G. Hevesy 
From the Institute of Theoretical Physics, University of Copenhagen 

We have administered labelled (radioactive) sodium phosphate to goats 
and investigated to what extent phosphorus present in different com- 
pounds extracted from the blood and the milk became labelled. In two 
cases the goat was killed after the experiment and the phosphorus 
compounds present in the organs investigated as well. Some of the 
results obtained are seen in the accompanying table. 



Activity per mgm P in milk. (Activity of 
plasma inorg. P after 414 boars taken as 1) 


Activity per mgm phosphatide P extracted 
from milk and organs, after 4}4 hours 


Interval after 

the start of the 

experiment 


Fraction 


Activity per 
mgm P 


Fraction 


Activity 
per mgm P 


0—2 hr.. 
2—41/2 hr. 


Inorg. P 
Casein P 
Ester P 

Inorg. P 
Casein P 
Ester P 

Inorg. P 
Casein P 
Ester P 

Inorg. P 
Casein P 
Ester P 


0.68 
0.54 
0.32 

1.79 
1.71 
1.16 

1.71 
1.71 
0.34 

0.49 
0.55 
0.49 


Milk 

Plasma 

Corpuscles 

Milk gland 

Liver 

Kidney 


0.09 
0.02 
0.01 
0.13 
0.09 
0.11 


41/2-61/2 hr. 


Activity per mgm P of milk 
ester P accumulated in — 3 
hours 




Hydrol. 7 min 0.76 


23—26 hr. 


Hydrol. 60 min 0.68 




Remaining fraction .... 0.34 




Milk, inorg. P 1.48 



Inorganic phosphorus 

The inorganic phosphorus extracted from the milk produced in the 
first two hours after the subcutaneous injection of the labelled phos- 
phorus, shows considerable radioactivity. Should the milk contain 



FORMATION OF MILK 305 

only those inorganic phospliorus atoms which were located in the plasma 
at some time after the start of the experiment, the specific activity 
of the milk inorganic phosphorus should be as high as that of the plasma 
inorganic phosphorus. In making such a comparison, it must be borne 
in mind that the specific activity of the plasma inorganic phosphorus 
rapidly decreases with increasing time through interaction of plasma 
phosphate phosphorus with that of bone and other tissue. No definite 
conclusion can therefore be drawn from comparing a single value of 
the specific activity of plasma and milk phosphorus. By following up, 
however, the change of the specific activity of the plasma inorganic- 
phosphorus and milk inorganic phosphorus with time, we find that i1 
takes 3 — 4 hours for the milk inorganic phosphorus to be almost entirely 
composed of individual atoms which had been present in the plasma 
after the start of the experiment. 

In milk produced shortly after the start of the experiment, a large 
part of the phosphorus atoms present were those which were located 
in the milk gland when the labelled phosphorus was administered. 
The replacement of the gland inorganic phosphorus by plasma inorganic 
phosphorus is thus comparatively slow because of a slow rate of penetra- 
tion of the phosphate ions through the cell walls. Heavy water, on the 
other hand, injected simultaneously with the labelled phosphate was 
already, after a short time, equally distributed between plasma and 
milk, because of the low resistance water molecules encounter when 
penetrating through cell walls. 

Casein phosphorus 

The comparatively high specific activity of the casein phosphorus is 
only compatible with the assumption that the phosphorus atoms utili- 
zed in the synthesis of the casein in the milk gland are drawn from the 
inorganic phosphorus of the plasma. From the difference in the rates 
at which the active casein phosphorus and the active inorganic phos- 
phorus present in the milk are formed, the time of formation of the 
casein in the gland cells can be estimated to be about 1 hour. 

Ester phosphorus 

The rate of formation in the milk gland of the average labelled phos- 
phorus ester molecule is lower than that of the average casein molecule 
(cf. table). II/4 hours after the administration of radioactive hexose- 
monophosphate (kindly presented to us by Prof. Parxas) injected into 
the veins of the goat, an appreciable amount of labelled ester was found 
in the milk, while another larger part of the activity was found in the 
inorganic milk phosphate. This result shows that a rapid enzymatic 

20 Hevesy 



306 ADVENTURES IN RADIOISOTOPE RESEARCH 

breakdown of the hexosemonophosphate and rebuilding of ester mole- 
cules takes place in the gland. The milk gland contains thus enzymes 
having the same action on hexosemonophosphate as Robison and 
Kray's^ bone extracts; however, the bulk of the esters present in the 
milk are acted on by enzymes present in the gland at a much slower 
rate. Similar behaviour is shown by the mixture of phosphorus esters 
present in the bloocP. 

Phosphatide phosphorus 

The formation of active phosphatide molecules is, as seen from the 
table, a slow process. The individual phosphatide molecules present in 
the milk were mainly built up in the milk gland and not taken up as 
such from the plasma (as is the case with the yolk phosphatide). This 
follows from the fact that the specific activity of the phosphatide phos- 
phorus extracted from the milk gland and also from the milk itself 
is higher than that secured from the phosphatide of the plasma. The 
view is often encountered that the milk fat originates from the plasma 
phosphatides which decompose in the milk gland, supplying fat and 
inorganic phosphorus. This view is entirely incompatible with the results 
obtained by us. To mention only one argument, we find the phosphatide 
phosphorus of the milk to be slightly, the inorganic phosphorus present 
to be strongly, active. The latter can therefore only originate from the 
highly active inorganic phosphorus of the plasma. 

It is well known that different milk fractions, secured consecutivel}' 
within a short time, have a markedly different fat content. As we find^ 
that the inorganic phosphorus extracted from these fractions has a 
different specific activity, we have to conclude that these fractions 
cannot originate from an initially homogeneous liquid. So we arrive 
at the result that some of the milk gland cells give off milk much more 
readily than others, but that some even of the first-mentioned cells 
retain a large part of their solid milk constituents, particularly the 
phosphatides (and fats). Not only are phosphorus compounds present 
in the milk not formed during the act of milking, as often assumed, 
but such compounds contained in the last fraction secured during the 
act of milking are partly of earlier date than those present in the im- 
mediately preceding milk samples. 

References 

L. Hahn and G. Hevesy Nature 140, 1059 (1937). 

R. RoBisoN The Significance of Phosphoric Esters in Metabolism (New York, 
1932). 

^ A detailed account of the experimental results obtained will be found in the dis- 
sertation of A. W. Aten, jun., to be presented to the University of Utrecht. 



307 



Comment on papers 27—33 

In 1935, after mailing paper 16 to Nature we embarked on the study whether 
and to what extent the constituents of the brain are renewed during adult life. 
After the administration of ='-P to rats an appreciable incorporation of the tracer 
into brain phosphatides was observed after the lapse of 1 lir or more (paper 27). 
These results were pubhshed simultaneously with those of Artom et al. (1937) 
and of Chaikoff et al. (1937) who demonstrated the incorporation of ^sp ji^to 
the phosphatides present in a great number of organs. We subsequently concen- 
trated our interest on the origin of phosphatides. Among the most fascinating 
apphcations of isotopic tracers ranges the study of the origin of body constituents. 
With LuNDSGAARD wc fed dogs with oil containing labelled sodium phos- 
phate in order to find out whether an appreciable part of the increased lecithin 
content in the blood is built up in the intestine was labelled (paper 28). These 
experiments showed that intestinal mucosa is not the chief place of sjTithesis 
of plasma phosphatides. It was the results of perfusion experiments (paper 29) 
which first indicated that the hver releases labelled phosphatides to the circula- 
tion. In other experiments (paper 30) not the removal of the labelled phospha- 
tides- from the hver but the uptake of these from the circulation by the liver was 
followed. These were the first experiments in which blood containing in vivo syn- 
thetized labelled compounds was transfused. They led to the result that not only 
is the rate of turnover of phosphatides in the liver very high, but the exchange 
of phosphatide molecules between the hver cells and the plasma takes place at 
a much higher rate than the corresponding process between other organs and the 
circulation. The hver was found to be the main source of formation of plasma 
phosphatides. This was most spectacularly demonstrated by Chaikoff and his 
group (1946) who in the course of their very extensive and important studies on 
phosphatide metabohsm have shown that in a hepatectomized dog, after administ- 
ration of labelled phosphate, the formation of labelled plasma phosphatides prac- 
tically ceases. In animals almost devoid of the higher unsaturated acids there is no 
diminution in the phosphatide turnover in the liver. An enhanced turnover rate is 
observed in the muscles of fat -starved rats (Hevesy and Smedley-Maclean, 
1940). 

That the chick builds up its own phosphatides and does not avail itself of the 
phosphatides in the yolk could be concluded from the following observation. 
After injecting labelled phosphate into the fertilized egg, the phosphatides extrac- 
ted from various tissues of the chick were strongly radioactive while the jolk 
phosphatides remained inactive (paper 32). 

That the phosphatide molecules of the milk in contrast to those of the yolk 
do not originate in the blood plasma but the former are built up in the milk gland 
could easily be proved (paper 33). The specific activity of the milk phosphatide 
phosphorus was found to amount to almost four times the specific activity of the 
plasma phosphatide phosphorus, but was lower than the corresponding value 
of the milk gland phosphatide phosphorus. The experiments were carried out at 
a time following administration of labelled sodium phosphate to the hen in which 
the activity of the plasma phosphatides was still increasing. In such experiments 
a milk phosphatide phosphorus specific activity which is higher than the corres- 
ponding value of the plasma phosphatides, excludes the plasma phosphatides 

20* 



308 ADVENTURES IN RADIOISOTOPE RESEARCH 

as a main source of milk phosphatides. A detailed description of these and a great 
number of additional experiments are described in the D. Sc. thesis of A. H. W. 
Aten Jr. (1939). 



References 

C. Artom, C. Perkier, M. Santangello and E. Segre (1934) Nature 139, 836. 

I. Perlman, S. Ruben and I. L. Chaikoff (1934) J. Biol. Chern. 122, 169. 

A. H. W. Aten Jr. (1939) Isotopes and Formation of Milk and Egg. Dissertation, 

Utrecht. 
C. Entenman, I. L. Chaikoff and D. B. Zilversmit (1946) /. Biol. Chem. 160, 5. 
G. Hevesy and I. Smedley-Maclean (1940) Biochem J. 34, 903 . 



Originally published in Kgl. Danske Vidcnskabeimes Selskab. Biologiske 

Meddelelmr. 15, 5 (1940) 

34. TURNOVER OF LECITHIN, CEPHALIN, 
AND SPHINGOMYELIN 

G. Hevesy and L. Hahn 

From the Institute of Theoretical Physics and Institute of Physical Chemistry, 

University of Copenhagen 

Phosphatide molecules present in tlie body have been taken up with 
the food or have been built up in the organism. A spectacular proof 
of the synthesis of phosphatides in the body is given by the fact thai 
ducks raised in diets containing phosphorus only in inorganic form laifl 
85 — 195 eggs during the summer^i\ These eggs contained 200 — 400 gm 
phosphatides (corresponding to 8 — 16 gm phosphatide P), and this very 
appreciable amount was synthesised by the organs of the clucks. On the 
other hand, phosphatides can enter the circulation from the intestine. 
The amount of phosphatide which is daily led by the intestinal lymph 
into the circulation of the rabbit^^^ on normal diet was calculated to be 
about 50 mgm. This is only about 1/5 of the amount daily synthesised 
in the liver (comp. p. 323); one must further consider that an 
appreciable part of the above mentioned 50 mgm was synthesised in the 
mucosa of the small intestine. Thus, the phosphatide molecules of the 
organs will be only to a small extent obtained directly from the food, 
the overwhelming majority being built up in the body. 



CONCEPT OF TURNOVER 

The ultimate aim of the investigation of the origin of Ww ])hosphatide 
molecules present in the body is to be able to state in which form ihr 
hydrogen, carbon, nitrogen, oxygen, and phosphorus atoms present 
in the phosphatide molecules were taken up by llic body and in what 
steps they were involved until ultimately incorporalcMl iiilo ))liosphatidc 
molecules. This exacting task can hardly l)e solved at prcscnl , ;\.vn\ we 
must content ourselves with the deteimination oC 1 he place and rale 
of formation oflhe phosphatide^ iiiolcculcs in 1 he body IVoiii glycerol. 



(i)G. FiNGERLiNG, Biochem . Z. 38, 44S (l<)ll). 

(2)H. SiJLLMANN and W. ^^■ILBRA^•^T, Biochem. Z. 270, 52 (1934) 



310 ADVENTURES IN RADIOISOTOPE RESEARCH 

fatty acid, choline (or another organic base), and phosphate. We will 
denote, in what follows, as turnover rate the rate of synthesis of phos- 
phatide molecules from inorganic phosphate and other components 
independent of the actual mechanisms involved, and we shall measure 
this rate by determining the extent to which labelled phosphate present 
in the cells of an organ is incorporated into these newly formed phos- 
phatide molecules. As the phosphatide content of an organ is usually 
constant, we can follow that with the formation of new phosphatide 
molecules the decomposition of an equal or similar number of old mole- 
cules goes hand in hand. The possibility must also be envisaged that new- 
formation and decomposition of phosphatides do not take place in the 
same organ, but that the newly formed molecules are synthesised in 
one organ and carried into the other by the circulation. 

The turnover rate can also be measured by following the rate of incor- 
poration of fatty acids or of choline, for example, into the phosphatide 
molecule. The turnover rates measured by using different indicators 
need not necessarily be identical. It would be conceivable, for example, 
that the incorporation of the phosphate radical into the phosphatide 
molecules w^ould be preceded by the formation of glycerophosphate and 
that this process would be a comparatively slow one in contrast to all 
other steps involved in the synthesis of the phosphatide molecule. 
In this case, the turnover rate measured, using labelled P as an indi- 
cator, would be slower than that found when using labelled fatty acids 
or labelled choline. The opposite would be the case if the reorganisation 
of the phosphate bond were to take place at a faster rate than the corre- 
sponding release and incorporation of fatty acids or choline into the 
phosphatide molecules. 

The question if and to what extent the rate of phosphate incorporation 
into the phosphatide molecule differs, for example, from that of the fatty 
acid incorporation into the latter cannot be answered at the time being. 

Feeding cats with mixed glyceride, the acids of wdiich were composed 
to 85 per cent of elaidic acid, Sinclair^i^ found 12 hours later the plasma 
phosphatide fatty acids to contain 19 per cent of elaidic acid. In our 
experiments we found (comp. p. 326) that, after the lapse of 16 hours, 
about 4 per cent of the phosphatides extracted from the plasma of rabbits 
contained labelled phosphate. 



(i)R. G. Sinclair, J. Biol. Chem. 115, 215 (1937). 



TURNOVER OF LECITHIN, CEPHALIN AND SPHINGOMYELIN 311 

INDICATORS APPLIED IN TURNOVER MEASUREMENTS 

a) Change of the degree of unsaturatioii of fatty acids 

Since the phosphatides contain both saturated and unsaturated fatty- 
acids, the change of the composition of the fatty acids of the organ 
phosphatides after ingestion of cod liver oil, for example, can be utilised 
to get information on the rate of the phosphatide turnover in the organ 
in question. A change in the iodine number of the phospholipids extracted 
from the liver of dogs^i^ and cats^^) after the ingestion of cod liver oil and 
the disappearance of the changes within 24 hours and 2 to 3 days, re- 
spectively, was observed at an early date. 

b) Incorporation of iodized fatty acids into the phosphatide molecule 

Iodized fatty acids, whether injected intravenously or given by mouth, 
enter the phosphatides of the liver, the blood^3)and the milk^^) for example, 

c) Incorporation of elaidic acid into the phosphatide molecule 

This method was repeatedly used in the investigation of the turnover of 
phosphatides. The rate of entrance of elaidic acid into and disappearance 
from the phosphatides was found to be rapid in the liver and the intesti- 
nal mucosa and comparatively slow in the muscle. The process was found 
to be essentially complete in the liver within a day, but in the muscle 
only after the period of many days(^). 

d) Incorporation of fatty acids, labelled by introduction of heavy hydrogen, 
into the phosphatide molecule 

Linseed oil was deuterated and the "heavy" fat obtained fed to rats. 
The investigation of the deuterium content of the phosphatides extracted 
from different organs gives information on the phosphatide turnover 
in the organ in question^. 



(1^ G. loANNOwics and E. P. Pick, Wien. Klin. Wochenschr. 23, 573 (1910), 

(2^ R. G. Sinclair, J. Biol. Chem.S2, 117 (1929). Comp. also B. G. Sinclaib, 
Phys. Rev. 14, 351 (1934). 

^3^ C. A. Artom, Arch. int. Physiol. 36, 191 (1933); C A. Artom and G. Peretti, 
Arch. int. Physiol. 36, 351 (1933). 

(*^ F. X. Aylward, J. H. Blackwood and J. A. B. Smith, Biochem. J. 31, 
130 (1937). 

(5>R. Sinclair, J. Biol. Chem. 111,270 (1935); 121, 161 (1937), M. F. Kohl, 
J. Biol. Chem. 126, 709 (1938). 

(6) B. Cavanagh and H. S. Raper, Biochem. J. 33, 17 (1939). 



312 ADVENTURES IN RADIOISOTOPE RESEARCH 

e) Incorporation of analogues of choline, in which arsenic replaces 
nitrogen, into the phosphatide molecule 

Arsenic can be detected in the lecithin fraction isolated from the 
liver and the brain of rats kept for 21 days on a diet containing arseno- 
choline chloride^^). 

f) Incorporation of labelled phosphate into the phosphatide molecule 

This method will be discussed in detail. 

Most of the methods outlined above were successfully applied to show 
that a marked turnover takes place in some of the organs, and the appli- 
cation of the methods a), c), and f) lead to the result that the rate of the 
phosphatide turnover is much faster in the intestinal mucosa and in 
the liver than in the other organs. None but the "phosphate method" 
was applied, however, to arrive at quantitative data as to the rate of 
rejuvenation of the phosphatide molecules present in the different 
organs. 



QUANTITATIVE DETERMINATION OF THE TURNOVER RATE BY 

USING LABELLED PHOSPHATE 

The formation of phosphatide molecules containing ^^p inside the 
tissue cell can only take place when the process of phosphatide formation 
was preceded by a penetration of ^^p into the cell, and the same applies 
to all indicators used in turnover experiments. This point was hitherto 
not considered. Its great importance is best seen by the following. 

To arrive at a proper figure for the turnover rate we have to compare 
the percentage of ^^p in the total inorganic P of the cells with the per- 
centage of ^2p in the total phosphatide P extracted from them. If these 
ratios, which correspond to those of the specific activities of the inorganic 
P and the phosphatide P, are found to be equal, we can conclude that 
all phosphatide molecules were renewed during the experiment. In this 
case, a proportional partition of ^^p between the inorganic P and the 
phosphatide P present in the cells took place. This is only possible if 
the phosphate radical of all the phosphatide molecules was split off in 
the course of the experiment, a process which was then followed by a 
synthesis of phosphatide molecules with incorporation of other phosphate 
radicals in which ^^pQ^ was represented proportionally to its total 
number present. If the specific activity of the phosphatide P is found, 



(i^A. Welch, Proc. Soc. Exp. Biol. Med. 35, 107 (1937). 



rURNOVER OF LECITHIN, CEPHALIN AND SPHINfiOMYKLIN 313 

to be, for example, lu per cent of that of the inorganic F, \v(^ can conclude 
that 10 per cent of the phosphatides were renewed during the experi- 
ment. 

Due regard must, however, be given to the change of the specific 
activity of the cellular inorganic P in the course of the experiment. 
By administering the labelled phosphate in several portions of suitably 
varying quantities in the course of the experiment, we can maintain 
a constant specific activity of plasma and interspace phosphate- As to 
the cellular concentration of ^^P, which is nought at the start of the 
experiment and then gradually increases, we determine the change of 
concentration with time experimentally and compare the specific acti- 
vity of the phosphatide P extracted at the end of the experiment with 
the average value of the specific activity of the inorganic P which pre- 
vailed during the experiment. 

When determining the specific activity of the cellular inorganic P^ 
due regard must be taken to the fact that a part of the tissue inorganic 
32P is of extracellular origin. As the extracellular volume of the tissue 
is known and the specific activity of the extracellular P does not differ 
much from that of the plasma P, we can easily correct for the presence 
of the extracellular P in the tissue inorganic P. Since the extracellular 
phosphate in the case of the muscle tissue, for example, amounts to 
only about 1/90 of the cellular inorganic P, the correction mentioned 
above becomes only significant in experiments of short duration. If the 
rate of penetration of the inorganic phosphate differs greatly in the cells 
of different tissues, as it actually does, for example, in the case of the 
liver and the muscle, we do not get proper information on the relative 
rate of turnover of the phosphatides in these organs by comparing the 
specific activity of the liver phosphatide P with that of the muscle 
phosphatide P. Conclusions based on such a comparison will greatly 
underestimate the relative rate of phosphatide turnover going on in the 
muscle cells into which the inorganic P diffuses at a slow rate, in contrast 
to its penetration into the liver cells. We will arrive, however, at correct 
figures by comparing the ratio. 

specific activity muscle phosphatide P 
specific activity muscle inorganic P 

with the corresponding ratio of liver products. 

If we wish to draw quantitative conclusions from experiments carried 
out with elaidic acid as an indicator, we have to compare the elaidic 
acid content of the organ phosphatides with thai of the elaidic acid 
content of the fatty acid mixture present in the corresponding cells in 
freely dispersed state. The latter magnitude is not known and the same 
consideration applies 1o the work with douterated fa1 as an indicator. 



314 



ADVENTURES IN RADIOISOTOPE RESEARCH 



We may get some, though very restricted, information by comparing 
the heavy hydrogen (D) content of the organ phosphatides with that 
of the organ glycerides. After the lapse of 10 hours, the ratio 

liver phosphatide D kidney phosphatide D 



liver glyceride D 



kidney glyceride D 



where D denotes the relative heavy content of the total "non-exchange- 
able" hydrogen, was found to be 1 : 2. 



EXPERIMENTAL PROCEDURE 

The labelled phosphate of negUgible weight, dissolved in physiological sodium 
chloride solution, was injected into the vena jugularis of the rabbit drop by drop 
during the experiment. Per hour 2.5 ec. were injected; the experiment took usually 




Fig. 1. Change of the specific activity of the plasma inorganic P 
during continuous intravenous injection of labelled phos- 
phate to a rabbit. (Specific activity = per cent of the labelled P 
injected, found in 1 mgm. P). 



4 hours. By taking small samples from the ear vein at different intervals, the 
change in the activity of the plasma was followed. In several cases, we extracted 
the inorganic P of the plasma and measured its specific activity (activity per mgm 
P), in others we contented ourselves with the measurement of the total activity 
of the plasma which, in experiments of short duration, is solely due to the inorga- 
nic phosphate present. 

The labelled P was injected drop by drop into the vena jugularis in order to 
obtaLa a comparatively small and easily accountable change in the activity level 
of the plasma (see Fig. 1). If aU the labelled P is injected at the start of the experi- 
ment, as in our early experiments and in all experiments carried out by other 
workers with labelled P, the activity level of the plasma is very high at the begin- 
ning, and it is slow at the end of the experiment (see Fig. 2). If the labelled P is 
given by subcutaneovis injection or by mouth, the activity of the plasma first 
increases with time and later decreases (see Fig. 3). The sensitiveness of the radio- 



TURNOVER OF LECITHIN, CEPHALIN AND SPHINGOMYELIN 



315 



3- 



FiG. 2. Cbange of the specific activity 
of the plasma inorganic P after sub- 
cutaneous injection of labelled phos- 
phate to a rabbit. 




mm. 



\ 



4-1 



Q. 

■a 



Fig. 3. Change of the logarithm 
of the labelled P content of the plas- 
ma with time after intravenous in- 
jection of labelled phosphate. 



= 



£1 



C71 
O 



50 



100 



150 



200 



Hours 



316 



ADVENTURES IN RADIOISOTOPE RESEARCH 



active indicator, thus, changes very appreciably in the course of the experiment. 
If we are successful in keeping the activity level of the plasma constant during 
the experiment, we can eliminate great difficulties otherwise encountered when 
calculating the turnover rate of organic phosphorus compounds. 

The changes in the activity of the plasma, shown in Fig. 1, can be further 
reduced by injecting amounts decreasing with time. In our later experiments 
we have chosen this procedure and varying amounts of labelled P were adminis- 



0,15 - 



.^0,10 



o 
o 



0,05- 




TT 



150 



200 



250 



mm. 



Fig. 4. Change of the specific activity of the plasma inorganic P 

during continuous subcutaneous injection of labelled 

phosphate to a rabbit. (Specific activity = per cent of the labelled 

P injected, found in 1 mgmP). 



tered by subcutaneous injection. In an experiment taking 12 hours, for exainple, 
labelled P was injected every 20 min. In experiments taking several weeks, in 
the later phases of the experiment injections were made twice a day. The change 
in the plasma activity in such an experiment taking 4 hours is seen in Fig. 4. 
In experiments taking several hours or days a constant activity level could be 
easily obtained. 

The determination of the turnover rate of the phosphatides present in the 
different organs necessitates the determination of the specific activity of the 
inorganic P and phosphatide P extracted from the organ. This determination 
was carried out in the following way. At the end of the experiment the animal 
was killed by bleeding. The organs were at once placed in Hquid air, minced, 
and extracted with cold 10 per cent trichloracetic acid. The inorganic phosphate 
present was precipitated as ammonium magnesium phosphate at 0°. Muscle samples 
were taken before death. To secure the phosphatide present in the organs, these 
were first dried with cold acetone and then treated with ether, later Avith boiling 
alcohol. The ether-alcohol extracts were evaporated in vacuo and taken up seve- 
ral times with petrol-ether; the phosphatides were then converted into phosphate 
by wet ashing. The procedure appHed when isolating lecithin, cephahn, and sphin- 
gomyelin will be discussed on page 330. 



TUIliS'OVEK OF LECITHIK, CKi'JlALIlS' AND srillNCiOM VKLIN 317 

CALCULATION OF THE TURNOVER RATE 

In most of our experiments only a minor pari of the phospliatide 
molecules present in the organ became labelled; we can, therefore, con- 
sider the reaction leading to the formation of labelled phosphatides to 
be a one-sided one and disregard the decomposition of lal)elled phospha- 
tides during the experiment. As already mentioned on page 312, to arrive 
at the value of the rate of the phosphatide turnover, we have to compare 
the specific activity of the phosphatide P extracted from the organ at 
the end of the experiment with the average specific activity of the cellu- 
lar inorganic P found in the course of the experiment. The value of the 
activity of the cellular inorganic P is obtained from that of the tissue 
inorganic P after subtraction of the share due to the extracellular fluid. 
The correction to be applied for the presence of extracellular P in the 
tissue inorganic P is, in most cases, a small one. In the liver of the rabbit, 
for example, out of 30 mgm. inorganic P only about 0.6 mgm is located 
in the interspaces. We arrive at this figure by assuming that the inter- 
spaces make out^^^ 22 per cent of the weight of the liver and the inorganic 
P content of the interspaces is 3 mgm per cent. The specific activity 
of the liver extracellular P is, after 4 hours, 2.5 times higher than the 
specific activity of the tissue inorganic P; correspondingly, 5 per cent 
of the total inorganic P activity of the liver is due to extracellular P. 

In the case of the muscle, we arrive by an analogous consideration 
at the result that 25 per cent of the activity of the tissue inorganic P is 
of extracellular origin. The extent of the correction to be applied increases 
with decreasing length of the experiment, since in experiments of short 
fluration only a small amount of labelled P penetrates into the cells. 

With regard to the considerations stated above, one must recognise the 
possibility that some of the phosphorus which one identifies, even after 
the most careful experimental procedure, as inorganic P, was in fact 
present in the tissue in the form of very labile, not yet known, organic 
phosphorus compounds. Very labile P compounds of that kind, if pre- 
sent, would probably be in fast exchange equilibrium with the inorganic 
P present, and their presence would therefore not influence much the 
calculation given above. The labile P of adenyltriphosphoric acid comes, 
for example, very quickly into exchange equilibrium with the inorganic 
P of the tissues or the corpuscles; it is often permissible to replace the 
specific activity of the inorganic P by that of the above mentioned labile 
P. The behaviour of creatinephosphoric acid is discussed on page 325. 

When calculating the turnover rate of phosphatides, we must consider 
the average specific activity of the cellular inorganic P prevailing during 
the experiment. This value is obtained by determining the specific 

(1) J. F. Manery and B. Hastings, J. Biol. Chem. 127, 657 (1939). 



318 



ADVENTURES IN RADIOISOTOPE RESEARCH 



activity of the tissue inorganic P and the plasma inorganic P at diffe- 
rent intervals. The change of the specific activity of the tissue inorganic 
P is seen in Table 1, that of the plasma inorganic P is discussed on 
page 315. 

Table 1. — Specific Activity of the Organ 

Inorganic P as Percentage of that of the 

Plasma Inorganic P 



Organ 


100 min 


240 miu 


Liver 


12.7 

0.8(1) 
14.7 
0.32 

85 


42.1 


Muscles 

Intestinal mucosa 

Brain(2) 

Kidneys 


4.6 
42.8 

1.4 
90 



<') In spite of all precautions taken, some creatine P may have been split off before the extraction of the 
inorganic P. The creatine P being, in experiments of short duration, less active than the inorganic P, such 
a decomposition may partly be responsible for the low value obtained in the experiment taking 100 min only. 

") Comp. p. 336 

It is of interest to remark that, in the case of the kidneys, after the 
lapse of 100 min an almost proportional partition of ^^p between plasma 
and cellular P is reached. When investigating, after 4 hours, the inorganic 
P of the marrow of the kidney, which makes out only a minor part 
of the total inorganic P of the kidney, the specific activity was found 
to be only 48 per cent of that of the plasma. 



CELLULAR AND EXTRA -CELLULAR FORMATION OF 

PHOSPHATIDES 

The turnover rates recorded in the fourth column of Tables 3 to 9 are 
calculated on the assumption that the formation of phosphatide molecu- 
les takes place inside the cells with participation of cellular inorganic P. 
Let us assume for a moment that the formation of phosphatide molecules 
takes place on the cell wall facing the interspaces. Then, not the cellular 
but the extracellular phosphate radicals^!) would enter the newly formed 
phosphatide molecules. As the specific activity of the extracellular 
inorganic P is often much higher than that of the cellular inorganic P, 
in the last mentioned case more active P atoms would take part in the 
synthetic process than in the first mentioned one. A high activity of 
the newly formed phosphatide would then not indicate such a high 



(1) From this view-point, it is without any significance whether the phosphate 
radical is directly incorporated into the phosphatide molecule or through inter- 
mediary stages. 



TURNOVER OF LECITHIN, CEPHALIN AND Sl'lllNGOMYELIN 



319 



turnover as it would if the formation of the phosphatide molecules took 
place with participation of the less active cellular P. It is obvious that 
the sensitivity of our radioactive indicator will be very different in the 
two cases mentioned above. Though it is much more probable that the 
turnover of the phosphatide molecules takes place inside the cells we 
have also recorded, in the fifth column of the above mentioned tables, 
the turnover rates calculated on the assumption of an extracellular for- 
mation of the phosphatide molecules. The values thus obtained give the 
lower limit of the turnover rate, while those obtained in column 4 give 

Table 2. — Specific Activity of the Inorganic P and Phosphatide P Extracted 

FROM the Organs 

Rabbit I. — Weight: 2.4 kgm 
Tiitraveiions injection during 4 hours 



Fraction 



Specific activity 
in relative units 



Plasma inorganic P 

Liver tissue inorganic P at the end of the experiment 

Liver tissue inorganic P corrected for the change in plasma activity 

during the experiment 

Liver cellular inorganic P at the end of the experiment corrected as 

above 

Liver cellular inorganic P average value during the experiment . . 

Liver phosphatide P 

Kidney tissue inorganic P at the end of the experiment 

Kidney tissue inorganic P corrected for the change in plasma activity 

during the experiment 

Kidney cellular inorganic P at the end of the experiment corrected as 

above 

Kidney cellular inorganic P average value during the experiment 
Kidney phosphatide P 



100 
36.2 

44 

40.8 

20.4 

3.0 

67.7 

82.3 

82.0 

73.5 

5.5 



the upper limit. It is conceivable that some of the phosphatide molecules 
are renewed inside the cell wall. In that case the inorganic P entering the 
newly formed phosphatide molecules will have a specific activity being the 
intermediary betw^een that of the extracellular and the cellular P. A conti- 
nuous drop of the specific activity of the inorganic P in the cell wall 
may therefore take place while the phosphate penetrates from the inter- 
spaces into the cells. 

In the corpuscles the phosphatides are known to be practically con- 
centrated in the stroma^^), andthe thickness^^) of the latter to correspond 



(1) B. N. Ebickson, H. H. Williams, S. S. Beknstein, J. Arvin, R. L. Jones 
and J. G. Macy, J. Biol. Chem. 122, 515 (1938). 

(2) Danielli, J. Cell. Comp. Physiol. 7, 393 (1936). 



320 



ADVENTURES IN RADIOISOTOPE RESEARCH 



to that of a few molecular layers. It is, therefore, quite conceivable 
that in the outer layer of the stroma a slow rejuvenation of the phos- 
phatide molecules takes place with incorporation of plasma P. In the 
case of finding an organic P fraction extracted from the cells or the 
corpuscles to show a higher specific activity than the cellular, respecti- 
vely corpus cular inorganic P, we would be justified to conclude that the 
synthesis of the organic compound in question did not take place inside 
the cells, respectively the corpuscles. Investigations in the above men- 
tioned direction may bring forward results of histochemical interest. 



RESULTS OF EXPERIMENTS 

Investigation of the total petrol-ether soluble phosphatide mixture 
Experiments with rabbits 



Table 3. — Spkcific Activity of the Cellular Inorganic P and Phosphatide 

P Extracted from the Organs 

Rabbit II. — Weight: 2.6 kgm 
Intravenous injection during 215 min 





Specific activity 


I'ercentage of phosphatides 
renewed daring the experi- 


Organ 


Inorganic P 
average during 
the experiment 


Phosphatide P 

at the end of the 

experiment 


ment 




A(') 


B(2) 


Liver 

Kidney 

Small intestine 


100 
382 
111 

.58 

.-)7.2 

66.3 

70.2 

40.8 


19.0 
18.3 
7.9 
4.46 
1.53 
4.04 
3.65 
1.63 


19.0 
4.8 
7.1 
7.7 
2.7 
6.1 
5.2 
4.0 


3.86 

3.7 

1.61 


Stomach 


0.91 


Heart 

Lungs 

Spleen 


0.31 

0.82 
0.74 


Marrow*'' 


0.33 


Brain 




0.06 





<'> Calculated on the assumption that the formation of phosphatides took place with incorporation of 
fellular inorganic P. 

(''' Calculated on the assumption that the formation of phosphatides took place with incorporation of 
extracellular inorganic P. 

(=' In several experiments the specific activity of the marrow inorganic P was found to be surprisingly 
low. even lower than that of the ester P. These low values were presumably due to the presence of traces 
of only slightly active bone P in tlie marrow sample. 

Critical remarks 

In Tables 2 — 9, data were given for the turnover rate of phosphatides 
in different organs of the rabbit. When calculating those values we 
assumed that the labelled phosphatides present in the organs were 
synthesised in situ. In the following, we will discuss how far this assump- 
tion is justified. 



TURN'OVER OF LBCITHI.V, CEPHALIX AXD SPHINGOMYELIN 



321 



Table 4. — Specific Activity of the Cellilar Inorganic P .\nd Phosphatide 

P Extracted fro.m the Organ.s 

Rabbit III. — Weight: 2.3 kgm. 
Iixtraveiious injection (luring 234 miii 





Specific activity 


Percentage of pliosphatides renewed 


Or?an 


Inorganic P 
averajje during 
the experiment 


Phosphatide P 
at the end of 
the experiment 


during the experiment 




AC) 


B(») 


Liver 

Muscles 


100 

7.8 


16.3 
0.56 


16.3 
7.2 


3.2 
0.11 



(») Turnover rate calculated on the assumption that the formation of phosphatides took place with 
incorporation of cellular inorganic P. 

'-=' Turnover rate calculated on the assumption that the formation of phosphatides took place witli 
incorporation of extracellular inorganic P. 

Table 5. — Specific Activity of the Cellltlar Inorganic P and Phosphatidh 

P Extracted from the Organs 

Rabbit IV. — Weight: 2.5 kgm. 
Intravenous injection dviring 215 min 





Specific activity 


Percentage of phosphatides renewed 


Organ 


Inorganic P 
average during 
the experiment 


Phosphatide P 
at the end of 
the experiment 


daring the experiment 




A(0 


B(»> 


Liver 


100 

374 

107 
64.6 
76.1 


14.8 

23.2 

20.0 
3.47 
7.67 
0.175 


14.8 
6.2 

18.7 
5.37 

10.1 


2.9 


Kidney 

Small intestine (mucosa) 

Heart 

Lungs 

Brain 


4.6 
3.9 

0.68 
1.51 



"' Turnover rate calculated on the assumption that the formation of phosphatides took place with 
incorporation of cellular inorganic P. 

»^' Turnover rate calculated on the assumption that the formation of phosphatides took place witli 
incorporation of extracellular inorganic P. 

Liver phosphatides 

Let us first consider the liver phosphatides. Apart from the liver, 
an intense turnover is going on in the intestinal mucosa, and the possibi- 
lity must be envisaged that the labelled phosphatides were carried 
into the liver from the intestine by the plasma. The plasma was founrl 
to contain only small amounts of labelled phosphatides, the specific 
activity of the plasma phosphatide P being, after the lapse of 4 hours, 
only 1/7 of that of the liver phosphatide P. This fact excludes the possi- 
bihty that a substantial part of the labelled liver phosphatides was led 
from the intestine or any other organ into the liver. Large amounts 



21 Hevesv 



322 



ADVENTURES IN RADIOISOTOPE RESEARCH 



Table 6. — Specific Activity of the Cellular Inorganic P and Phosphatide 

P Extracted from the Organs 

Rabbit V. — Weight: 2.1 kgm 
Intravenou.s injection during 2.50 min 





Specific 


activity 


Percentage of phosphatides renewed 


Organ 


Inorg.anio P 
average during 
the experiment 


Phospliatide P 
at the end of 
tlie experiment 


daring the experiment 




Ad) 


B(2) 


Liver 


100 


18.6 


18.6 


2.76 


Kidney 


364 


22.8 


6.3 


3.58 


Small intestine (mucosa) .... 


115 


23.6 


20.5 


3.54 


Muscle 


12.0 


0.87 


7.3 


0.11 



(•) Turnover rate calculated on the assumption that the formation of phosphatides took place with 
incorporation of cellular inorganic P. 

(-) Turnover rate calculated on the assumption that the formation of phosphatides took place with 
incorporation of extracellular inoi'ganic P. 

Table 7. — Specific Activity of the Celli'lar Inorganic P and Phosphatide 

P Extracted from the Organs 

Rabbit VI. — Weight: 2.6 kgm 
Subcutaneous injection during 255 min 





Organ 


Specific activity 


Percentage of phosphatides renewed 




Inorganic P 
average during 
the experiment 


Phosphatide P 
at the end of 
the experiment 


during the experiment 




A.M B<2) 


Liver 


100 

29.0 


14.8 
1.51 


14.8 
5.2 


3.2 


Corpuscles 


0.33 



<') Turnover rate calculated on the assumption that the formation of phosphatides took place with 
incorporation of cellular inorganic P. 

C') Turnover rate calculated on the assumption that the formation of phosphatides took place with 
incorporation of extracellular inorganic P. 



of water can be led from one pond into the other by a narrow channel; 
salt water, however, (salt corresponding to labelled phosphatides in 
our case) cannot pass the channel without the water of the channel 
becoming salt as well. The concept of "specific activity" proves, thus, 
to be of great use when putting forward considerations such as those 
discussed above. 

One may say, in respect of these considerations, that, while the specific 
activity of the average plasma phosphatides is low, one of the phos- 
phatide fractions (phosphatides represent a mixture of numerous com- 
pounds) might be synthesised at a very fast rate in the intestinal mucosa, 
and the labelled molecules formed in this process might have rushed 
through th(> plasma at a last rale inio Ihe liver without raising much 



TURNOVER OF LECITHIN, CEPHALIN AND SPHINGOMYELIN 



323 



T.\BLE 8. — Specific Activity of the Cellular Inorganic P .\nd Phosphatide 

P Extracted from the Organs 

RabVjit VII. — Weight: 2.4 kgm. 
Siibciitaneoiis injection during \l.r> honi-s 





Specific activity 


Percentage of phosphatides renewed 


Organ 


Inorganic P 


Phosphatide P 


during tlic experiment 




average during 


at tlio end of 








the experiment 


the experiment 


Ad) 


B(») 


Liver 


100 


25.2 


25.2 


14.9 


Corpuscles 


25.5 


4.03 


15.8 


2.39 


Muscles 


14.7 


1.31 


8.9 


0.78 


Brain 


36.53 


0.55 
31.8 




Marrow 


87.0 


18.8 



<') Turnover rate calculated on the assumption that the formation of phosphatides took place with 
incorporatiou of cellular inorganic P. 

<") Turnover rate calculated on the assumption that the formation of phosphatides took place with 
incorporation of extracellular inorganic P. 

*'> As the presence of traces only of 'cone Pin the marrow sample investigated lowers the specific activity 
of the marrow inorganic P, the recorded figure for the inorganic P of the marrow may be too low and that 
recorded for the rate of renewal of the phosphatide P of the marrow, correspondingly, too high. 



Table 9. 



Extent of Renewal of Phosphatides 



Rabbit IX. — Weight: 2.5 kgm. 
Subcutaneous injection during 50 days 





Organ 


Tercentage of jjhospl 
not renewed 


latides 




AM 


B(2) 


Liver 




73 



3 







Muscle 

Marrow 


64 



Corpuscles 


3 



<') Rate of renewal calculated on the assumption that the formation of phosphatides took place with 
incorporation of cellular inorganic P. 

(*) Rate of renewal calculated on the assumption that the formation of phosphatides took place with 
incorporation of extracellular inorganic P, 

the specific activity of the average plasma phosphatide P. As shown 
on page 340, the specific activity of the phosphorus present in different 
phosphatide fractions can differ substantially, but, in spite of exhaustive 
fractionation processes no fraction of extremely high or extremely low 
specific activity was found. Furthermore, the total amount of labelled 
phosphatides formed in the intestinal mucosa in the course of 4 hours 
amounts to only 1/5 of that formed in the liver during the same time. 
In this connection it is of interest to remark that, according to the 
results obtained by Sullmann and Wilbrandt which are discussed 
on page 309, the intestinal lympli carries up to 0.1 mgm phosphatide 



21* 



324 ADVENTURES IN RADIOISOTOl'E RESEARCH 

P<^) per hour; but, even if this amount of newly formed phosphatides is 
quantitatively led from the intestine into the liver, it would not suffice 
to account for the presence of the amount of newly formed phospha- 
tides found in the latter which corresponds to more than 0.5 mgm 
phosphatide P per hour. 

An entirely different argument against the intestinal origin of the 
labelled phosphatides found in the liver is the following. The labelled 
phosphatides present in the plasma were not found to leave the blood 
stream at a very fast rate, half of the labelled phosphatides present 
leaving the plasma in the course of an hour, 30 per cent of these phospha- 
tides being found in the liver^^) thus, a rapid rush of labelled phosphatides 
through the plasma does not take place. 

That the labelled phosphatides found in the liver are, at least to a large 
extent, formed in situ, was also shown in experiments on isolated perfused 
liver Such investigations were formerly^^) carried out by us on isolated 
cat livers in which, after the lapse of 2.5 hours, the specific activity of 
the liver phosphatide P was found to he about 1.5 per cent of that of 
the liver inorganic P. A further proof that the phosphatides present 
in the liver were formed there was brought about by Chaikoff and his 
colleagues(^) who found that, in experiments on rats, the removal of 
tissues very active in phospholipid turnover, namely the gastrointestinal 
tract and the kidneys, does not markedly influence the phospholipid 
Uirnover in the liver. 



Muscle phosphatides 

After discussing the origin of the labelled liver phosphatides we shall 
put forward similar arguments as to the origin of the labelled muscle 
phosphatides. The specific activity of the plasma phosphatides is found 
to be about 3 times higher after the lapse of four hours than that of 
the muscle phosphatides. Considerations based on the comparison of 
the specific activity of the plasma phosphatides and the muscle phospha- 

*'' When oil is fed to the rabbit, twice that amount was found to be carried by 
the intestinal lymph. The feeding of oil raises the rate of turnover in the intestinal 
mucosa and the liver as well, as shown in experiments on rats (C. Artom, G. 
Sabzana and E. Segre, Arch. Int. Physiol. 47, 245 (1938); B. A. Fries, S. Ruben, 
J. Perlman and J. L. Chaikoff, J. Biol. Chem. 123, 587 (1938) and also on isola- 
ted peifused cat liver, where the turnover rate was fotmd to be about twice as 
high as in experiments in which non-lipemic (normal) blood was used (L. Hahn 
and G. Hevesy, Bicchem. J. 32, 342 (1938). 

(2)L. Hahn and G. Hevesy, Nature 164, 72 (1939). 

(3)L. Hahn and G. Hevesy, Biochem. J. 32, 342 (1938). 

(*^ B. A. Fries, S. Ruben, J. Perlman and J.L. C'nMKorF, J. Biol. Chem. 
123, 567 (1938). 



TURNOVER OF LECITHIN, CEPHALIX AND SPHINGOMYELIN 325 

tides do not, therefore, exclude the possibility that the labelled phospha- 
tides present in the muscles were carried into them from other organs. 
This possibility is, however, excluded by the result of experiments based 
on the rate of entrance of labelled phosphatides into the muscles<i). 
While, in the course of 4 hours, phosphatides showing a relative activity 
of 0.54 units pass from the plasma into the muscles, phosphatides having 
an activity of 160 units were found 1o be present in the muscles after the 
lapse of the same time. 

In experiments of short duration Ihe creatine P of the muscles gets 
only partly labelled and, therefore, a decomposition of creatinephos- 
phoric acid prior to the extraction of the inorganic P will lead to a 
■"'dilution" of the activity of the inorganic P present as such in the muscle 
tissue. The possibility that in our experiments, taking only a few 
hours, too low values are obtained for the specific activity of the 
muscle inorganic P cannot, therefore, be entirely discarded. As the 
extent of the new formation of the muscle phosphatides is calculated by 
comparing the specific activity of the phosphatide P with that of the 
inorganic P, a too low value of the specific activity of the inorganic P will 
manifestly lead to a too high value of the rate of new formation of the 
phosphatides. 

Kidney phosphatides 

Kidney phosphatide P is found in experiments of short duration to be 
more active than the phosphatide P extracted from all other organs. 
From this fact we may, however, not follow that the kidney phospha- 
tides are renewed at a faster rate than the phosphatides in the liver 
or the intestinal mucosa. The labelled inorganic P of the plasma diffuses 
with a remarkable speed into the kidney cells (see Table 1). This is 
in no way surprising in view of the role of the kidney cells as to excretion 
and re-absorption of phosphate. A result of this fast penetration of 
active phosphate into the kidney cells will be a formation of active 
phosphatide molecules already in the earliest stages of the experiment. 
This is not the case in the cells of such organs into which the labelled 
phosphate diffuses at a slower rate. 

Labelled phosphatides of the plasma 

The renewal of phosphatides in the plasma can only be determined in 
experiments in vitro; in such experiments,^-) taking 4.5 hours, the specific 



(i^L. HAHN-and G. Hevesy, Nature 144, 204 (1939); Kgl. Dansle Vidensk. 
Selskah, Biol. Medd. 15, G (1940). 

(2) L. Hahn and G. Hevesy, Mem. Carlsberg 22, 190 (1937). 



326 



ADVENTURES IN RADIOSIOTOPE RESEARCH 



activity of the plasma phosphatide P was found to be smaller than 
1/1000 of that of the inorganic P. 

In experiments in vivo, an exchange between plasma phosphatides 
and organ phosphatides takes place and, as in some of the organs labelled 
phosphatides are formed at a fast rate, we will soon after the administra- 
tion of labelled phosphate find labelled phosphatide molecules in the 
plasma, which were released from the organs. In fact, almost all phospha- 
tide molecules found in the plasma were synthesized in the organs. 
The labelled phosphatide content of the plasma, at different times, is 
seen in Table 10. In this experiment, the labelled inorganic P content 
of the plasma was kept constant during 9 days. 

Table 10. — Specific Activity of Phosphatide P and 
Inorganic P of the Plasma 



Time 



KeUitive specific activity 



Inorganic P Phosphatide P 



4 hours 
16 hours 
25 hours 
37 hours 
45 hours 
55 hours 

9 days 



100 
100 
100 
100 
100 
100 
100 



0.53 
3.8 
8.1 
15.0 
22.0 
27.5 
81.6 



Three consecutive processes have to precede the appearance of label- 
led phosphatides in the plasma. Labelled inorganic P has to diffuse into 
the cells of the liver and other organs in which the plasma phosphatides 
are formed. The building up of the labelled phosphatide molecules 
represents the second process, their release into the plasma the third. 
In view of the time taken by these processes, it is easy to understand 
that in the early stages of the experiment the change of the labelled 
phosphatide content of the plasma has a more rapid than linear depend- 
ence with time. 

Since a large part of the phosphatide molecules found in the plasma 
originated from the liver, it is of interest to compare the amount of the 
active phosphatides found in the plasma with that present in the liver 
at the end of the experiment. 

As seen in column 3 of Table 11, after the lapse of 12 hours, the acti- 
vity of the plasma phosphatides reached 3/4 of that of the liver phospha- 
tides. A large part of the liver phosphatides is, however, not yet renewed 
and a further substantial increase of the activity of the plasma phospha- 
tides can only be expected by a corresponchng increase in the active 
phosphatide content of the liver and other organs. 



TURNOVER OF LECITHIN, CEPIIALIN AND SPHINGOMYELIN 



327 



Table 11. — Active Phosphatide Content of the 
Liver anp the Plasma of Rabbits 







Extent of partition of 


Duration of the 


Ratio of active pliospliatide 


labellcil pliospliatiiles 


experiment 


content of liver and plasma 


between liver phosphatides 
and plasma phosphatides 


4 houi-s 


94 


O.K) 


12 hoiii-s 


18 


0.76 


9 days 


14 


1.0 



Phosphatide turnover in the corpuscles 

Compared with the phosphatide turnover going on in the organs, 
the phosphatide turnover taking place in the corpuscles is but little. 
This is also shown by results obtained when investigating the origin 



Table 12. — Extent of Partition of Labelled 

Phosphatides, Originally Present in the Pla.sma, 

between the Phosphatides of the Corpuscles and 

OF THE Plasma in Experiments in vitro 

(Plasma of a rabbit containing labelled phosphatides shaken 
with corpuscles of another rabbit) 



Animal 


Time in hours 


Extent of par- 
tition (Percen- 
tage) 


Rabbit ' 


0.5 
1.5 
3.0 
4.5 


1.8 
3.6 




4.0 
5.0 


Hen 


1.5 
2.0 
3.0 


1.5 
2.0 
1.5 



of the yolk phosphatides(i). In these experiments, 28 hours after admi- 
nistration of the labelled phosphate, the specific activity of the corpuscle 
phosphatides w^as found to be only 1/3 of that of the plasma phosphati- 
des; showing the corpuscles to be, so-to-say, a by-path of the liver and 
other organ phosphatides on the way through the plasma into the yolk. 
The labelled phosphatide molecules of the corpuscles have various 
origins. Some of them were incorporated in the course of the red cell 



(1^ G. Hevesy and L. Hahn, Kgl. Danske Vidensk. Selfkab. Biol. Medd. 14, 
2 (1938). 



328 



ADVENTURES IN EADIOISOTOPE RESEARCH 



formation into a tissue containing labelled phosphatides. Some of the 
labelled phosphatide molecules came into the corpuscles after they 
reached the circulation. As seen in Table 12, in which the results of 
some experiments in vitro are recorded, a part of the phosphatide 
molecules of the corpuscles exchanges easily with those of the plasma. 
Presumably those situated in the outermost layer of the stroma take 
part in this exchange process. It is, however, rather difficult to interpret 
the comparatively high specific activity of the phosphatide P extracted 
from the corpuscles in experiments in vivo without assuming that 
a phosphatide turnover takes place in the corpuscles, though the rate 
of this turnover is small compared with that of most of the acid-soluble 
P compounds present in the corpuscles (see Table 14). 



Table 13 — Extent of Partition of Labelled 
Phosphatides, Originally Present in the Plasma, 

BETWEEN the PHOSPHATIDES OF THE CoRPTJSCLES 
AND OF THE PlASMA IN EXPERIMENTS in vivO 









Extent of par- 


Animal 


Time 


in hours 


tition (Percen- 
tage) 






24 


16 


Rabbit (2— 2.5kgm) , 




24 

24 


18 
17 






25 


16 






42 


34 


Chicken (100— 150 gm) j 


18 
22.5 


6.0 

8.1 



In experiments in vivo with rabbits (see Table 13), in the course of a 
day, the activity of the corpuscle phosphatide P was found to be only 
about 1/6 of that of the plasma phosphatide P. A still greater difference 
was found when investigating chickens blood. 

Using elaidic acid as an indicator, Sinclair^i) found, 8 hours after 
ingestion of the elaidic acid, 15 per cent of the fatty acids extracted 
from the plasma phosphatides to be composed of this distinctive fatty 
acid, while the corpuscles contained no more than traces of the indicator. 

When iodised fatty acid was used as an indicator, it was found^^) not 
only in the phosphatides of the plasma but also in those of the cor- 
puscles. In the latter, the concentration of iodised fat was even higher 
(3.3 per cent of the 1otal fatty acids) than in the former (2.0 per cent). 
The application of iodised fatty acids leads, thus, to a result which is in 



(i>R. G. Sinclair, J. Biol. Chem. 115, 211 (1930). 
(2)C. Abtom, Arch. Int. Physiol. 36, 101 (1933). 



TURNOVER OF LECITHIX. CEPHALIX AND SPHINGOMYELIN 



320 



contradiction to that obtained by using labelled phosphate or elaidic 
acid as indicators. Phosphatides containing iodised fatty acids are 
possibly selectively taken up by the corpuscles, another explanation 
being that the molecules of these compounds present in the plasma were 
decomposed at a faster rate than those incorporated into the stroma. 
Phosphatides containing iodized fatty acids represent non-physiolo- 
gical compounds and, as shown by the above example, the results obtai- 
ned by using such indicators must be interpreted very cautiously. 

Table 14. >— Specific Acttvitv of Phosphatide P and 
Ac ID Soluble P of the Corpuscles 











KeUitive 


specitic 


activity 


F 


ruction 








after 






4 hours 


12 hours 


Phosphatide P'" . . 








2.6 






9.6 


Inorganic P 








100 






100 


Pyrophosphate P . . 








99.. 5 






100 


Hydrolyzed by 1 n 


H^SO, 


in 


7 to 100 min. 


100 




1 




Hydrolyzed in 100 


min to 


12 


hours 


100 






100 


Non-hydrolyzed . . . 








87 









<•> The active phosphatide molecules are partly such ones which were taken up from the plasma by a 
exchange process. 

In this connection, the observation^^) should be also mentioned that 
in lactating cows during fasting a marked decrease in the concentration 
of plasma P lipids takes place which persists for several weeks after reali- 
mentation, but there is no significant change in the amount of red cell phos- 
phatides. This result also shows the absence of an intense interaction bet- 
ween plasma phosphatides and phosphatides present in the corpuscles. 

PART II 

Investigation of lecithin, cephalin, and sphingomyelin 

We discussed above the rate of renewal of the average petrol-ether 
soluble phosphatide molecules; in the following, we wish to describe 
some experiments in which lecithin, cephalin, and sphingomyelin were 
separately investigated and their turnover rate determined. Chemically^ 
cephalin differs from lecithin by containing aminoethanol instead of 
choline. The biological consequence of this replacement is very signifi- 
cant(2). Cephalin is highly active in accelerating blood clotting, whereas 



(i)j. A. Smith, Biochem. J. 32, 1856 (1938). 

(2> Comp. E. Chajigaff, M. Ziff and B. M. Hogg, J. Biol. Chem. 131, 35 (1939).. 



330 ADVENTURES IN RADIOISOTOPE RESEARCH 

lecithin is not. It was even reported^^) that cephalin prepared from cattle- 
blood or cattle-brain enhances, while lecithin inhibits the clotting of 
rabbits blood. The role of the phospholipids as transport agents of fats 
was much discussed, this role being often ascribed to lecithin alone. 
In our first experiments, we determined the turnover rate of lecithin 
and cephalin in the organs of rabbits 4 hours after intravenous injection 
of labelled phosphate. We found the turnover rate of cephalin extracted 
from the liver, the intestinal mucosa and other organs to be pronoun- 
cedly faster than that of lecithin. Simultaneously, Chargaff^^) found 
the rate of rejuvenation of cephalin extracted from the liver and the 
intestinal tract of rats to be slower than that of lecithin. We were first 
inclined to explain this difference in the findings of Chargaff and 
ourselves by the fact that the former investigated the turnover process, 
in contradistinction to us, in carnivorous animals. We soon found, 
however, that it is the duration of the experiment which is decisive for 
the higher or lower rate found for the cephalin turnover. We will, in what 
follows, first describe the experimental procedure used. 

Experimental procedure 

The tissue is dried with cold acetone and extracted first with ether 
and then with boiling alcohol. The second process is repeated several 
times. The solutions obtained were evaporated to dryness and taken up 
by petrol-ether in the presence of pulverised dry sodium phosphate. 
The latter was added in order to remove traces of active phosphate 
possibly present. The process was then repeated in the absence of phos- 
phate and the dry residue taken up in ether. The next step was to pre- 
cipitate the cephalin from the solution by adding 96 per cent alcohol. 
The filtrate obtained was evaporated and the residue containing lecithin 
extracted with ice-cold alcohol. This procedure was repeated and the 
purified lecithin obtained precipitated as chloro-cadmium-lecithin. The 
compound obtained was thoroughly washed with ether in order to remove 
traces of chloro-cadmium-cephalin possibly present. 

The cephalin was prepared from the alcoholic precipitate obtained 
in the early treatment of the phosphatide mixture. To obtain pure 
cephalin the precipitate was repeatedly dissolved in ether and precipi- 
tated with alcohol. 

To secure sphingomyelin the fraction insoluble in petrol-ether was 
collected and treated alternatively with ether and ice-cold alcohol. 
The last residue thus obtained was dissolved in a mixture of methyl 
.alcohol and chloroform. By adding ether to this solution purified sphingo- 

^i^Y. Ok.\rmura, Mitt. med. Oes. Okoyama 48, 1585 (1936). 
^2)E. Chargaff, J. Biol. Chem. 128, 592 (1939). 



TURNOVER OF LECITHIN, CEPHALIN AND SPHINGOMYELIN 



3:}i 



myelin was precipitated. A further purification of this product was 
obtained by repeating the procedure described above. When sufficient 
amounts were available, the sphingomyeltn was recrystalUsed from pyridin. 

Experiments with rabbits 

In the experiments, the results of which are given in Tables 15 and 
16, all the labelled phosphate was administered at the start of the expe- 
riment. In all later experiments, labelled phosphate was administered 

Table 15. — Specific Activity of Inorganic P and of 
Different Phosphatide Fractions 
Rabbit X. — Weight: 2.9 kgm 
All labelled phosphate was administered at the start of the experi- 
ment by stomach tube. The animal was killed after 19 houi-s 





Specific activity relative to the 


Fraction 


Plasma 
inorg. P 


Inorg. P of 
the organ 




found at tlie end of the 
experiment 


Plasma lecithin P 

Liver inorganic P 

Liver lecithin P 


39.1 

89.7 
46.3 
35.4 
28.2 
4.67 
0.40 
1.04 


100 
51.6 


Liver cephalin P 

Liver spliingomyelin P 

Brain inorganic P 

Brain lecithin P 


39.5 
31.2 

100 
8.6 


Brain cephalin P 


22.4 



Table 16. — Specific Activity of Inorganic P and P of 
Different Phosphatide Fractions 
Rabbit XI. — Weight: 2.2 kgm 
Labelled phosphate administered to the rabbit by subcuta- 
neous injection at the start of the experiment. — The animal 
was killed after the lapse of 7 days 



Fraction 



Relative specific 
activitv 



Plasma lecithin P 

Plasma cephalin P 

Plasma sphingomyelin P 
Corpuscles lecithin P . . . 
Corpuscles cephalin P . . 

Brain inorganic P 

Brain lecithin P 

Brain cephalin P 



100 
48.1 
74..-) 
88.5 
73.1 
26.6 
14.1 
20.9 



332 



ADVENTURES IN RADIOISOTOPE RESEARCH 



all through the experiment to keep the specific activity of the plasma 
inorganic P at a constant level. In the experiments of short duration tak- 
ing only 4 hours, the cephalin extracted from all the organs investi- 
gated was found to be much more active than the lecithin. While the 
sphingomyelin extracted from the liver did not much differ in its speci- 
fic activity from that of the lecithin of this organ, in the muscle the 
sphingomyelin was found to be much more active than the lecithin but 
less active than the cephalin. 

In experiments taking 12 hours, lecithin and cephalin were renewed 
in the liver to about the same rate while sphingomyelin was found to 
show a slower turnover rate. The relative activity of lecithin and cepha- 
lin was, thus, very materially different in the experiment taking 12 
hours from that found in experiments of only 4 hours duration. This 
is not the case with the different phosphatide fractions secured from 

Table 17. — Renewal of Lecithin and Cephalin 

Rabbit III. — Weight: 2.3 kgm 
Intravenous injection during 234 min 



Fraction 


Percentage of pliospliatide renewed 
during the experiment 




av') 


B(2) 


Liver lecitliin 


10.9 
27.9 


2.1 


Liver cephalin 


5.5 



<•> Turnover rate calculated on the assumption that the formation of phosphatides took place with 
incorporation of cellular inorganic P. 

<^' Turnover rate calculated on the assumption that the formation of phosphatides took place with incor- 
poration of extracellular inorganic P. 



Table 18. — Renewal of Lecithin, Cephalin and Sphingomyelin 

Rabbit IV. — Weight: 2.5 kgm 
Intravenous injection during 215 min 





Percentage of phosphatides renewed during the experiment 


Organ 


A<') 


B<2) 




Lecithin 


Cephalin 


Sphingo- 
myelin 


Lecithin 


Cephalin 


Sphingo- 
myelin 


Liver 


J4.38<*M 
|3.67<''/ 

17.0 


26.5 
40.6 


4.4 


f 0.86>'> ] 
\ 0.72('>j 

3.55 


5.2 

8.47 


0.86 


Small intestine 

(mucosa) 





<'^ Turnover rate calculated on the assumption that the formation of phosphatides took place with 
incorporation of cellular inorganic P. 

^^1 Turnover rate calculated on the assumption that the formation of phosphatides took place with 
incorporation of extracellular inorganic P. 

<'> Fraction extracted with cold ether (not protein-bound lecithin?). 

*') Fraction extracted, after removal of the ether-soluble lecithin, with hot alcohol (protein-bound?). 



TURNOVER OF LECITHIN, CEPHALIN AND SPHINGOMYELIN 



:3:3:i 



the muscles. In these fractions, both after 4 and after 12 hours cephaUn 
and sphingomyelin are more active than lecithin. 

When looking at the results of the experiments taking one day or 
more (see Tables 15, 16 and 22) we notice that the lecithin extracted 
from the liver is more active than the cephalin, while the opprjsite was 
found to be the case for the fractions secured from the brain. 

Table 19. — Renewal of Lecithin, Cephalin and Sphingomyklix 
Rabbit V. — Weight: 2.1 kgm 
Intravenous injection during 250 min 





Percentage of phosphatides renewed during the experiment 


Organ 


Ad) 


B(0 




Lecithin 


Cephalin 


Sphyngo- 
myelin 


Lecithin 


Ceplialiu 


Spliingo- 
myelin 


Liver 


1.4 
3.7 

( 2.9<^' 1 
1 1.6'" j 

15.6 


24.6 
13.5 

21.7 
33.4 


8.95 
15.1 


1.86 

2.10 
f0.044">| 
|0.024'^'j 

2.69 


3.68 

7.7 

0.33 
5.77 


1.34 


Kidney 

Aluscle 


23 


Small intestine 

(^mucosa^ 









<'' Turnover rate calculated on the assumption that the formation of phosphatides took place with 
incorporation of cellular inorganic P. 

'■''> Turnover rate calculated on the assumption that the formation of phosphatides took place witli 
incorporation of extracellular inorganic P. 

*^> Fraction extracted with cold ether. 

(5) Fraction extracted, after removal of the ether-soluble lecithin, with hot alcohol. 

Table 20. — Renewal of Lecithin and Cephalin 
Rabbit VI. — Weight: 2.6 kgm 
Sxibcutaneous injection during 255 nain 



Fraction 



Percentage of phosphatides renewed 
during the esperiment 



A(0 



B(=) 



Liver lecithin . 
Liver cephalin 




2.2 
7.9 



I'' Turnover rate calculated on the assumption that the formation of phosphatides took place witli 
incorporation of cellular inorganic P. 

'^' Turnover rate calculated on the assumption that the formation of phosphatides took place with 
incorporation of extracellular inorganic P. 



Discussion 



The fact illustrated by the results described above —that in experi- 
ments taking only a few hours, the cephalin shows a higher extent of 
renewal than the lecithin, while, in experiments taking one day or more, 
the opposite is the case — suggests that not all cephalin present in the 



334 



ADVENTURES IN RADIOISOTOPE RESEARCH 



Table 21. — Renewal of Lecithin, Cephalin and Sphingomyelin 
Rabbit VII. — Weight: 2.4 kgm 
Subcutaneous injection during 11.5 hours 





Percentage o£ pliosphatides renewed during the experiment 


Organ 


Ad) 


B(«) 




Lecitliin 


Cephalin 


Sphingo- 
myelin 


Lecithin 


Cephalin 


Sphingo- 
myelin 


Liver 

Muscle 


/27.o(3) \ 

(20.0(4) 1 

5.6 


25.9 
20.6 


14.8 
17.2 


fl6.3(3)l 
|13.4(4)j 
0.49 


15.3 
1.81 


8.8 
1.51 







<i) Turnover rate calculated on the assumption that the formation of phosphatides took place with 
incorporation of cellular inorganic P. 

("> Turnover rate calculated on the assumption that the formation of phosphatides took place with 
incorporation of extracellular inorganic P. 

(3) Fraction extracted with cold ether. 

<^) Fraction extracted, after removal of the ether-soluble lecithin, with hot alcohol. 



Table 22. — Renewal of Lecithin, Cephalin 
AND Sphingomyelin 
Rabbit VIII. — Weight: 2.0 kgm 
Subcutaneous injection during 9 days 



Fraction 


Specific activity 
at the end of 
the experiment 


Lower limit of 

percentage 

renewed 


Liver inorganic P 

Liver lecithin P 


100 
84.0 
84.8 
40.3 
18.7 
12.5 
11 
16.1 
18.8 
17.3 
5.3 
5.6 
100 
82 
94 
61.7 


84.0 
84.8 


Muscle inorganic -f- creatine P 

Muscle ester P 

Muscle lecithin P 

Muscle cephalin P 

Muscle sphingomyelin P 

Brain inorganic P 

Brain ester P 

Brain lecithin P 

Brain cephalin P 

Plasma inorganic P 

Plasma phosphatide P 

Corpuscle acid soluble P 

Corpuscle phosphatide P 


46.4 
31.0 
27 
40.0 

92 
28 
30 

65.6 



organs is renewed at the same rate, some fractions showing a much faster 
turnover rate than others. These fractions could differ either in their 
chemical composition or in their location in the cells. Numerous chemi- 
cally different cephalins and lecithins exist differing, for example, in 
the type of fatty acids they contain. It is, however, not probable that 
the difference in the chemical constitution is responsible for the remark- 



TURNOVER OF LECITHIN, CEPHALIN AND SPHIN(;OMVRTJN 



335 



able difference in the turnover rate of the different cephahn fractions. 
The specific activity of successive fractions of cephalin (^rystalHsed 
repeatedly from alcoholic or other solutions docs not vary appreciably 
(comp. p. 341). A much more probable explanation of the difference 

Tablk 23. — Renewal of Lecithin and Ckphalin 
Rabbit IX. — Weight: 2.. 5 kgm 
Subcutaneous injection during 50 daj'S 





Organ ' 


PorfPiltafji' of phosphatiiies rciinwed 
during the oxperiment 




Lecithin 


Cephalin 


Liver 

MaiTow 


100 

100 
7.5(1) (42(2)) 
74(1) (65(2)) 


100 
100 


Brain 


81(1) (4ti(2)) 
71(1) (62(2)) 


Muscle 





<'' Calculated on the assumption of formation inside the cells (with incorporation of plasma inorganic P). 
''' Calculated on the assumption of formation outside the cells (with incorporation of plasma inorganic P). 



20- 



10- 




12 Hours 
Fig. 5. Turnover of lecithin and cephalin in the liver. 



mentioned above is that in some parts of the cell a decidedly more 
pronounced enzymatic breakdown and building up of cephalin takes 
place than in others. In experiments of short duration, we mainly mea- 
sure the rejuvenation taking place in these favoured districts. The beha- 
viour of lecithin is different in that we do not encounter such a pronoun- 
ced variation in the rate of turnover of different fractions. The average 
lecithin molecule is, however, renewed at a similar rate as the average 
cephalin molecule. This explanation is suggested by the fact that, while 



336 



ADVENTURES IN RADIOISOTOPE RESEARCH 



in experiments of short duration tiie cephalin P extracted from the liver, 
for example, is found to be more active than the lecithin P, in experi- 
ments of long duration both fractions are found to have about the same 
activity. Not only cephalin and lecithin extracted from the organs 
of the rabbit show this behaviour, but also the phosphatide fractions 
secured from the organs of rats, frogs, hens, and of isolated cat liver. 




50 Days 
Fig. 0. Turnover of lecithin and cephalin in the brain. 

That, in the case of the muscle and brain tissue, cephalin is found 
in experiments of long duration as well to have a faster turnover rate 
Than lecithin is in no way in contradiction to the conclusion arrived 
at when investigating the liver fractions. All phosphatide fractions 
present in the muscle, and especially those in the brain, are renewed 
at a comparatively slow rate. This remark applies also to the "fast" 
cephalin fraction present which, though "fast" relative to the average 
cephalin or lecithin, is in fact "slow". This slowness has the effect that 
seven days do not suffice to reach the point where the amount of label- 
led lecithin is larger than that of the labelled cephalin. The considera- 
tions made above are iUustrated by Figs. 5 and 6. 



Brain phosphatide 

While, in the case of other tissues, the penetration of phosphate from 
the plasma into the interspaces can be considered as an almost momen- 
tary process and, accordingly, the specific activity of the plasma inor- 
ganic P can be taken to be equal to that of the extracellular inorganic P, 
It cannot in brain tissue. We find that after 4 hours only 1/3 of the amount 
of labelled phosphorus (inch organic P) is present in the brain tissue, 
which we should expect to be present in the extracellular space alone 
in case of a prompt distribution of the labelled phosphate between the 



TURNOVER OF LECITHIN, CEPHALIN AND SPHINGOMYELIN 



337 



plasma and the interspaces. The extracellular space of the brain tissue 
was calculated from the chlorine or sodium distribution to amount to 
30 per cent of the weight of the tissue<i\ 

Table 24. — Formation of Labellkd PHO.snrATini-s ix titf: Brain of thk KAumr 



Duration of the experiment 


Spec, activity 

of brain 

inorganic P 


Spec, activity 

of brain 
phosphatide P 


Spec, activity 

of brain 
phosphatide P 


Spec, activity 

of brain 
phosphatide P 


Spec, activity 

of plasma 

inorganic P 


Spec, activity 

of plasma 

inorganic P 


Spec, activity 

of brain 

inorganic P 


Spec, activity 

of liver 
phosphatide P 


215 mill 


0.013 

0.015 

0.030 

0.19 

0.56 


0.0001 

0.0002 

0.0033 

0.054 

0.43 


0.0093 

0.016 

0.11 

0.29 

0.77 


0.0032 


250 mill 


0.005 


1 1 ..5 lioui's 


0.022 


9 davs 


0.063 


50 days 


0.43 



In view of the foregoing statements, we cannot give exact figures 
for the specific activity of the extracellular and cellular inorganic P 
of the brain. Since these figures enter the calculation of the turnover 
rate of the brain phosphatides the calculation cannot be carried out. 
A further complication arises from the fact that the decomposition of 
the brain creatinephosphoric acid prior to the extraction of the inorga- 
nic P could not be avoided in our experiments. The brain creatine P 
may be appreciably less active than the brain inorganic P. This fact 
would lead to a dilution of the labelled inorganic P by non-labelled 
inorganic P. We record, therefore, in Table 24, a) the specific activity 
of the brain phosphatide P relative to the plasma inorganic P, h) relative 
to the brain total inorganic P, and c) relative to the liver phosphatide P. 
While the brain phosphatides are found to be much less active than the 

Table 25. — Formation of Labelled Lecithin and Cephalin 
IN the Brain of the Rabbit 



Kelative specific activity 



Duration of the experiment 

i 


Plasma 
inorganic P 


Brain 
lecithin P 


Brain 
cephalin P 


250 min 


100 
100 
100 
100 


0.0092 
0.25 
0.40 
42 




11.5 hours'" 


1.08 


19 hoursi 

50 days 


1.04 
46 



<•) In this case, the total activity was injected at the start of the experiment. 



<i^ J. F. Manery and B. Hastings, J. Biol. Chem. 127, 657 (1939). 



22 Hevcsy 



338 ADVENTURES IN RADIOISOTOPE RESEARCH 

liver phosphatides, high values are obtained for the ratio of the specific 
activity of the brain phosphatide P and the brain inorganic P. Even 
if v^^e divide these values by 2, to account for the diluting effect of the 
creatine^^^ P, the resulting figures will still be high. 

Changus, Chaikoff and Ruben<2) observed a progressive increase 
in the content of radioactive phosphatides in the brain on rats for about 
200 hours after the administration of labelled phosphorus and it is of 
interest to note that, in a recent investigation, Chaikoff and his 
colleagues(3) found that the specific activity of the phosphatide P is not 
uniform throughout the central nervous system. 

Experiments uith rat 

The specific activity of lecithin P and cephalin P expected from the 
rat's liver is given in Tables 26 and 27. 

While the ratio of the specific activity of cephalin and lecithin P was 
found, after 3 hours, to be 1.33, after 24 hours we find the value 0.7. 
Similarly Chargaff<'*^ found, in experiments taking 24 hours, greater 

Table 26. — Specific Activity of Lecithin 

AND Cephalin in the Rat's Liveb. 

Weight of the rat: 200 gm 

All labelled phosphate was injected subcuta- 

neously at the start of the experiment; 

190 min later, the rat was killed 



Fraction 


i'ercent of ai^tivity injec- 
ted, found ill 1 nigm 
phosphatide P 




0.21 
0.28 


Liver cephalin 



turnover figures for lecithin than for cephalin. He found the above ratio 
to be 0.8. It is also interesting to note that an early paper of Aetom 
and his colleagues contains data on the relative activity of lecithin and 
cephalin extracted from the liver of rats to which olive oil and labelled 
sodium phosphate was administered 9 hours previously. They state the 
above ratio to be about 0.6. 

(i^ C. Artom, C. Perkier, M. Santangello, G. Sarzana and E. Segre, Arch. 
Int. Physiol. 45, 35 (1937). 

^2>G. W. Changus, J. L. Chaikoff and S. IIjjbe^, J. Biol. Chem. 126, 493 
(1938). 

(3) B. A. Fries, G. W. Changus and J. L. Chaikoff, J. Biol. Chem. 132, 24 
(1940). 

(4>E. Chargaff, ./. liiol. Chem. 128, 592 (1939). 



TURNOVER OF LECITHIN, CEPHALIN AND SPHINGOMYELIN 



339 



Tablk 27. — Spkcikio Activity ok Dii-fehent P 

Fr.\ctions in thk Rat'.s Liver 

Weight of the rat: 222 gm 

All labelled phosphate was injected subcutaneoiisly 

at the start of the experiment; 24 houre later, the 

rat was killed 



Liver fraction 



Relative specific 
activity'"' 



Labile P . 

Lecithin P 

CephaUn P 

Non-labile acid-soluble P 
Protein P 



100 
117 

82 

103 

31 



<"' The figures are given relative to the labile acid-soluble P, the value of which, after 24 hours?, (;losely 
corresponds to that of the inorganic P. 

Experiments with frogs 

The turnover figures of eephalin and lecithin extracted from the frog's 
liver were found, as would be expected, to be lower than the correspond- 
ing figures found in experiments with mammalia. The specific activity 
of the eephalin P was found to be much higher than that of the lecithin P. 

Table 28. — Specific Activity of Different P 

Fractions in the Liver of a Frog 
Labelled phosphate was injected into the lymph-sack 
of a frog kept at 20° all through the experiment 

(4 hours) 



Liver fraction 


Relative specific 
activity 


Inorganic P 

Cephalin P 

Lecithin P 


100 
7.8 
1.3 







Ratio of specific activity of cephalin and lecithin = 6. 

Experiments with laying hens 

That, in a laying hen, the specific activity of cephalin P of the liver 
is found, after the lapse of 5 hours, to be slightly higher than that of 
lecithin P, while in experiments with rabbits a very great difference 
was found, is just the result which we have to expect in view of the 
arguments discussed on p. 335. In the course of 5 hours, the phosphatide 
molecules present in the liver of the hen are renewed to an extent which 
in the case of the rabbit is first reached after the lapse of many hours . 



22* 



340 ADVEXTURES IX RADIOISOTOPE RESEARCH 

Table 29. — Specific Activity of P Fractions 

IN THE Organs of a Hen Weighing 900 gm 
Labelled phosphate was administered to a laying hen 
by subcutaneous injection at the start of the expe- 
riment; the hen was killed 5 hours later 



Fraction 



Relative speciiic 
activity 



Plasma lecithin P 

Plasma cephalin P 

Liver lecitliin P 

Liver cephalin P 

Liver spliingomyelin P 

Liver protein P 

Kidney lecithin P 

Kidney cephalin P 

Intestinal mucosa lecithin P .... 
Intestinal mucosa cephalin P . . . . 
Intestinal mucosa sphingomyelin P 



1.00 
0.98 
2.76 
2.93 
1.38 
0.15 
L15 
1.69 
0.90 
1.05 
1.10 



It is, therefore, not surprising that the fractions obtained from the 
lien's liver are similar to those secured from the rabbit's liver in experi- 
ments of much longer duration. In the kidneys of the laying hen the 
phosphatide molecules are renewed at a slower rate than in the liver 
and, in this organ, as was to be expected, cephalin is found to be mark- 
edly more active than lecithin. 

The liver sphingomyelin of the laying hen which does not enter the 
yolk to any appreciable extent is renewed at a decidedly lower rate 
than the petrol-ether soluble phosphatides. It is also interesting to note 
that the rate of rejuvenation of the protein P in the liver of the laying 
hen is about 20 times slower than that of the phosphatide P. 

In the intestinal mucosa, cephalin and sphingomyelin are formed at a 
somewhat higher rate than lecithin. In the kidneys cephalin was found 
more active than lecithin. That the rate of renewal of phosphatides 
in the liver of laying hens is decidedly higher than in the intestinal 
mucosa or other organs was also found in our earlier researches. (i) 

Experiment with perfused cat liver 

The experiment on cat liver which was carried out with the kind help 
of Professor Lundsgaard also indicates the faster cephalin turnover 
in experiments of short duration. The fasting cat used in this experiment 
weighed 3.3 kgm. Blood circulated for 70 min through the isolated livcM-. 



1 G. Hevesy and L. Hahn, Kgl. Danske Vidensk- Selskab, Biol. Mcdd. 14, 
2 (1938). 



TURNOVER OF LECITHIN, CEPHALIX AND SPHINGOMYELIN 341 

Besides labelled phosphate of negligible weight, 500 mgm alcohol was 
added to the blood at the start of the experiment and 500 mgm glycine 
after 30 min. 

When fractionating the alcoholic solution of the liver cephalin, the less 
soluble fraction was found to show the higher specific activity amounting 

Table 30. — Specific Activity of P Fractions 

IN the Liver of a Cat 

Duration of experiment: 70 min. 



Fraction 


Relative specific 
activity 


Plasma inorganic P 

Plasma lecithin P 

Liver lecithin P 

Liver cephalin P 


100 
0.18 
2.43 
4.05 



Ratio of tlic .activity of cephalin P and lecithin P = 1.67. 

to 4.47. The low lecithin activity of the plasma is, in view of the short 
duration of the experiment, not surprising. The labelled phosphate 
requires some time to penetrate into the liver cells, the formation of 
labelled lecithin takes some time as well and, finally, the release of the 
phosphatides into the plasma is far from being a momentary process. 

Survey of the results 

In the course of 4 hours, an appreciable part of the petrol-ether soluble 
phosphatides present in the intestinal mucosa and the liver were found 
to be renewed. This result is in conformity with that found by Artom 
and his colleagues(i), by Chaikoff and his collaborators^^), and in this 
laboratory<3). In Tables 31 and 32, a summary of the data obtained on 
the renewal rate of lecithin, cephalin and sphingomyelin fractions is 
given. In Table 32, the very different behaviour of lecithin from cephalin 
is clearly seen. While, in the case of lecithin, the labelled percentage 
increases more or less linearly with time, this is far from being the case 
with cephalin. We find an almost linear increase with time in the amount 
of labelled lecithin formed in the liver and the muscles, assuming that 
the formation of this compound takes place inside the cells. This linearity 
does not hold if w^e assume the formation of phosphatides to take place 
with incorporation of extracellular P. The bulk of the labelled liver 

^1^ C. Artom, C A. Perrier, M. Santangello, G. Sarzana and E. Segre, 
Arch. Int. Physiol. 45, 32 (1937). 

(2) B. A. Fries, S. Ruben, J. Perlman andJ. L. Chaikoff, J. Biol. Chem. 
123, 587 (1938). 

(3)L. Hahn and G. Hevesy, Nature 144, 204 (1939). 



342 ADVENTURES IN RADIOISOTOPE RESEARCH 

Table 31. — Extent or Renewal of the Petrol-ether 
Soluble Phosphatide Mixture -Extracted from the 

Organs of the Rabbit in the Course of 4 Hours 
The results are computed from the figures of Tables 2 — 7. 



Organ 



Percentage of phosphatides 
renewed during the experiment 



AC) 



B<*) 



Small intestine (mucosa) 19.6 3.7 

Liver 16.7 3.1 

Lungs 8.1 1.2 

Stomach 7.7 i 0.9 



Muscle 7.3 0.11 

Kidney 6.2 4.3 

Spleen 5.2 0.74 

Corpuscles 5.2 0.33 

Heart 4.0 0.50 

t" Turnover rate calculated on the assumption that the formation of phosphatides took place with 
ncorporation of cellular inorganic P. 

<'i) Turnover rate calculated on the assumption that the formation of phosphatides took place with 
ncorporation of extracellular inorganic P. 

lecithin could not be formed in the last mentioned way, since in that 
case (see column 5 of Table 32) nine times as much labelled lecithin 
should have been formed in the course of 12 hours than was found after 
4 hours. Similar considerations apply to the muscle lecithin where in 
the course of 12 hours seventeen times as much labelled lecithin should 
have been formed as after 4 hours. Such an increase with time is highly 
improbable. 

If we consider the two possibilities of the formation of cephalin, i. e. 
incorporation either of cellular or of extracellular labelled inorganic P, 
we arrive at the following result. If the labelled cephalin is formed inside 
the liver cells, as much as 1/4 became labelled within 4 hours; thus, 
1/4 of the total cephalin present undergoes a rapid renewal, the remain- 
ing 3/4 being comparatively inert. In the course of the following 8 hours, 
hardly any further increase of the amount of newly formed cephalin 
can be noticed. That the remaining part of the cephalin is also renewed, 
though at a very slow rate, is, however, shown by the fact that, after 
9 days, most of the cephalin present at the start of the experiment 
was found to be labelled. Muscle cephalin behaves in an analogous way. 

If we now consider the possibility that the labelled cephalin is formed 
with incorporation of extracellular P, we arrive at an entirely different 
interpretation of the results. The amount of labelled liver cephalin 
formed in the course of 12 hours then works out to be about three times 
that formed duiing 4 hours. This result is quite plausible. The result 
obtained in the case of the muscle cephalin, where as much as five times 
more labelled cephalin should have been formed in the course of 12 



TURNOVER OF LECITHIN, CEPHALIN AND sriilNGOirVELIN 



343 



Table 32. — Extent of Renkwal of LKirrmN, Cephalin and Sphingomyklin 
IN THE Organs of the Rabbit ix the Course of Experiments Lasting 4 Hours 

AND 12 HorRS, Respectively 

The results are computed from the Tigures in Taliles 17 — 21. 



Orpin 



rercentage of phosphatides reuewed 



.\(" 



Lecithin 



Oeplinlin 



Sphingo- 
rayelin 



I!(=) 



Lecitiiin 



Ccpliiiiin 



Sphinpo- 
niyelin 









after 


4 houi-s 






Small intestine 














(mucosa) 


16.3 


37 


— ■ 


3.1 


7.1 


— 


Liver 


9.3 


28 


(i.7 


1.7 


5.6 


1.1 


Muscle 


2.3 


21 


15.1 


0.03 


0.33 


0.23 


Kidney 


3.7 


13 





2.1 


7.7 


— 









after 12 hours 






Liver 


25 


26 


15 14.6 


15.3 


8.8 


Muscle 


5.6 


21 


17 0.5 

1 


1.8 


1.5 



<•) Turnover rate calculated on the assumption that the formation of phosphatides took place with 
incorporation of cellular inorganic P. 

<*) Turnover rate calculated on the assumption that the formation of phosphatides took place with 
incorporation of extracellular inorganic P. 

than in 4 hours, seems less plausible. While it is not probable that the 
cephalin present in the liver should have been formed with incorporation 
of extracellular inorganic P, we must envisage the possibility that a 
part of the cephalin located in the cell membranes is renewed with 
incorporation of inorganic P located inside the membrane. We discussed 
above the two extreme cases, formation of phosphatides with incorpo- 
ration of cellular and of extracellular P. While penetrating through 
the cell wall, the inorganic P may experience a more or less continuous 
drop in its activity and the renewal of phosphatide molecules located 
in the cell membranes could take place by incorporation of "intermediary" 
labelled phosphate radicals. 

It is of interest to remark that in a laying hen, where the liver has 
to supply large amounts daily of both lecithin and cephalin, the "slow" 
cephalin fraction is also renewed at a remarkable rate and the rejuven- 
ation of the average lecithin and cephalin in the course of 5 hours hardly 
differs. 



Difference between "fast" and "slow" cephalin 

That the organs contain a small cephalin fraction which is renewed 
at a fast rate and a larger one which is slowly renewed may possibly be 
due to a difference in the chemical composition of these fractions. Since 



344 ADVENTURES IN RADIOISOTOPE RESEARCH 

different cephalin fractions obtained by fractional crystallisation of 
the total cephalin extracted from the organ in question did not show- 
large variations, it is not probable that the above result can be explained 
as due to different rates of new formation of cephalins of different chemi- 
cal composition. 

In fractional crystallisation of alcoholoc cephalin solutions, only 
minor differences in the specific activity of the fractions were noticed. 
The least soluble fraction extracted from the liver showed, for example, 
a turnover rate of 4.47, while the value found for the average fraction 
was 4.05. When organs were extracted first with ether and then with 
hot alcohol, the lecithin prepared from the first extract was found to 
be somewhat more active than was the lecithin prepared from the 
alcohol extract (see Table 19). 

Since the renewal of cephalin is an enzymatic process, its velocity 
should be determined by the effectivity of the enzymes present. It is 
probable that that part of the cephalin which is located in such a region 
of the cells, where the enzymatic action is very pronounced, is renewed 
at a very fast rate. It is also probable that this "fast" fraction has a 
different biological significance from the "slow" fraction. The fact that 
the phosphatides have a much larger turnover in some organs than in 
others induced Sinclair^^) to distinguish between metabolic and non- 
metabolic phosphatides. The former ones found in the liver, for example, 
should be involved in fat metabolism; the latter ones, found for example, 
in the muscle, should play an important role in building up cell membra- 
nes. Our results suggest the interpretation that we have in all the organs 
nvestigated a "fast" and a "slow" cephalin fraction as well. The "fast" 
fraction is the smaller one. To what extent the "fast" cephalin and other 
phosphatide fractions are involved in fat metabolism is under investi- 
gation. 

Summary 

Labelled sodium phosphate was administered to rabbits, rats, frogs and laying 
hens. In order to keep the concentration of the labelled phosphate in the plasma 
constant, labelled phosphate was injected from time to time throughout the 
experiments. 

The specific activity of the inorganic P extracted from the plasma and the 
organs was measured at intervals. From these data the average specific activity 
of the cellular inorganic P prevailing during the experiment was calculated. 

The phosphatides present in various organs were extracted as well, and the 
specific activity of th-^ f)hosphatide P and also of the lecithin, cephalin and sphin- 
gomyelin P determinod. 

The knowledge of the average specific activity of the cellular inorganic P 
during the experiment and that of the phosphatide P at the end of tht experimen^ 

^i^R. G. Sinclair, Physiol. Rev. 14, 357 (1934). 



TURXOVEK OF LECITHIN, CEl'HALIX AND SPniXGOMVElJX 345 

permits us to calculate the extent of new formation (tuinoxer) of the phosphati- 
des on the assumption that this process takes place inside the cells. In case 
the phosphatide molecules are renewed with incorporation of extracellular inor- 
ganic phosphate, the specific activity of the latter enters the calculation. 

The specific activity of cephalin P extracted from different organs was found 
in experiments of short duration (4 hours) to be much higher (up to 10 times)' 
than that of lecithin P. With increasing time of expeiiment this difference was 
found to diminish. In the fractions obtained from the rabbits liver, after the 
lapse of 12 hours, both fractions showed the same activity. In organs like muscle 
and brain, in which a slow phosphatide turnover takes place, an equal activity 
of lecithin and cephalin is only reached after the lapse of several daj-s. 

Sphingomyehn is renewed in the liver at a slower rate than the ether- soluble 
phosphatides. In the muscles, in experiments taking not longer than 12 hours, 
sphingomyehn was found to be appreciably more active than lecithin, but less 
active than cephahn; after the lapse of 9 days, sphingomyelin was found to be the 
most active fraction. 

Two alternative explanations are put forward to explain the difference in the 
behaviour of cephalin and lecithin : (a) a part (about 1/4) of the cephalin present 
inside the cells is renewed at an appreciably higher rate than the average cephalin 
present, while the bulk of the cephalin showed a similar turnover rate as the ave- 
rage lecithin; or (b) a part of the cephalin located in the cell walls is renewed in 
situ with incorporation of inorganic phosphate which has a higher specific activity 
than the inorganic P located inside the cells. 

In the course of 50 days, all phosphatide molecules present in the Uver and 
the skeleton were found to be renewed. However, only 74 per cent of the lecithin 
and 71 per cent of the cephahn extracted from the muscles were newly formed 
in the course of the experiment. In the brain tissue, 1/4 or more of the lecithin 
and 1/5 or more of the cephalin molecules remained tinchanged. 

The amount of active lecithin and cephalin present in the plasma and corpuscles 
was determined. The active plasma phosphatide molecules are not formed in the 
circulation but in the organs and are led into the circulation. Most of the phos- 
phatide molecules present in the corpuscles were incorporated during the for- 
mation of the erythrocytes, but some turnover takes place inside the corpuscles^ 



Originally published in Acta Physiol. Scand. 19, 370 (1950) 

35. TURNOVER OF PHOSPHATIDES 

Grace db C. Elliott and G. Hevesy 

From the Institute for Research in Organic Chemistry and the Laboratorj- of 
Pharmacology of the Karolinska Institute, Stockholm 

Since the time when it was first shown that ^^p administered to animals 
is easily incorporated into liver phosphatides (Artom et al., 1937; 
Hahn and Hevesy, 1937; Perlman et al., 1937) and the first attempt 
to arrive at a quantitative figure for the rate of renewal (turnover) 
of liver phosphatides (Artom et al., 1938; Hevesy and Hahn, 1940 a), 
the problem of phosphatide turnover in the animal organism has found 
an ever-increasing interest. The above-mentioned observation was not 
surprising in view of the early observation by Artom (1933) that iodi- 
nated fatty acids, when administered, promptly enter the phosphatides 
of the liver, of Sinclair's (1936) similar observation on feeding elaidic 
acid, and the still earlier work by Ivanowics and Pick (1910) on the 
change in iodine number of phosphatides extracted from the livers of 
dogs after ingestion of cod li^'e^ oil. 

In later studies with ^^p^ liver phosphatides were found to be the 
almost sole source of plasma phosphatides^-^ to interchange readily 
between liver and plasma, and also to be metabolized to a large extent in 
the liver. Furthermore, numerous investigations were carried out with 
the aim to elucidate the effect on phosphatide formation by feeding 
choline, cholesterol, amino acid, etc. Surveys of these investigations 
were recently given by Chaikoff and Zilversmit (1948) and by Hevesy 
(1948). 

PRECURSOR OF PHOSPHATIDE PHOSPHORUS 

The application of isotopic indicators in turnover studies often aims 
at the determination of the average life-time of a type of molecules 
in an organ. We wish, for example, to know the time during which the 

^1^ Artom et al. (1948) demonstrated recently that fed intact labelled phospha- 
tide molecules can be absorbed into the circulation. These absorbed phosphatide 
molecules were found, however, to account for a minor part only of the total 
ainount of ^-P fed as phosphatide P. 



TUKNOVEH OF PHOSPUATIDES 347 

average phosphatide (or lecithine and so on) molecule in llu^ liver remains 
unchanged. The occurrence of a change may involve certain moieties of 
the molecule only or a reassembly of all the moieties or. finally. 1 he mole- 
cule may leave the organ unchanged, as do some of the phosphatide 
molecules of Ihe liver, which interchange with phosphatide molecules 
of the plasma. In the last mentioned case, the determination of the 
rate of renewal (replacement) of the phosphatides does not encounter 
difficulties. When replacing part of the plasma of one animal by plasma 
of another animal, containing ^ap labelled phosphatides, we can follow 
the rate of disappearance, thus the rate of replacement of plasma phos- 
phatides. In the first mentioned cases w(> ma} , however, encounter great 
difficulties due to our ignorance of the steps involved in the biosynthe- 
sis of most types of molecules. We can, however, determine the upper 
limit of the time during which the average phosphatide molecules of the 
liver, for example, remain unchanged by administering labelled phosphate 
and comparing the specific activity of the phosphatide P at the end of 
the experiment with the specific activity of the intracellular inorganic P 
which prevailed in the average during the experiment. In experiments 
of short duration, the repeated renewal of a phosphatide molecule may 
usually be neglected. Is that not the case, a more complicated calcu- 
lation has to be used, as will be shown below (cf. p. 360). 

The rate of incorporation of labelled intracellular orthophosphate 
may indicate the rate of renewal of the phosphate group of the phos- 
phatide molecule, it will, however, not necessarily do so. If first with 
the participation of orthophosphate, or with a phosphorus compound 
which comes into rapid exchange equilibrium with orthophosphate 
at a comparatively slow rate, a formation of phosphorus containing pre- 
cursor of the phosphatide molecule takes place, and this formation is 
followed by a relatively rapid incorporation of the precursor into the 
phosphatide molecule, the following up of the rate of incorporation of 
the intracellular orthophosphate into Ihe phosphatide molecules fails 
to reveal the rapid interchange taking place between \hv ullimate 
precursor and the phosphatide molecule. 

It is quite possible that the labelled orthophosphate is incorporated 
Into the phosphatide molecule during the asseml)ly oi ihe molecule 
and that thus orthophosphate or a phosphorus compound which comes 
into rapid exchange equilibrium with orlhophosphatc as ATP is not 
only an early, but also the last precursor of phosphatide P. It is, how- 
ever, equally possible that glycerophosphate or another organic P com- 
pound is the last precursor of phosphatide phosphorus. 

Following a single administration of labelled phosphate the specific 
activity of the liver intracellular orthophosphate first increases ; in the 
later phase of the experiment, however, when the decrease in specific 
activity of the plasma inorganic P results in a partial exodus of ^^p 



348 ADVENTURES IN RADIOISOTOPE RESEARCH 

from the liver cells into the plasma, the specific activity of the liver 
orthophosphate P decreases. While, in the first phase of the experiment, 
with increasing time an increasing incorporation of ^ap takes place 
into liver phosphatides, in the second phase of the experiment, the 
already labelled phosphatide molecules are continually renewed and, 
as this renewal takes place in a medium of decreasing activity, the spe- 
cific activity of the phosphatide P also decreases. The slower the orga- 
nic compound is labelled in the first phase of the experiment, the slower 
it will lose its activity in the second phase, as was already found in 
early experiments with phosphatides and will be discussed below. 
A precursor will thus have a higher specific activity than the product 
formed in the early phase of the experiment and a lower one in the 
second phase. To these two criteria an important third was added by 
ZiLVERSMiT and assoc. (1943), viz. that the maximum specific activity 
of the precursor P must coincide with the beginning decrease in specific 
activity of the phosphatide P. Often the practical application of this 
"Zilversmit criterium" encounters difficulties. Based on cumulative 
evidence including specific activity-time relations, Zilversmit and 
assoc. (1948) arrive, however, to the result that it is likely that glyc- 
erophosphate is the pertinent precursor of liver lecithin. 

We shall in the following mainly consider the rate of incorporation of 
orthophosphate, which has the same specific activity as the labile 
phosphate of ATP, into phosphatides. Even though the consideration 
that orthophosphate P is the precursor of phosphatide P, leads to a 
lower limit of the turnover rate only, it involves many advantages 
which one appreciates when faced with the task to calculate the turn- 
over rates in investigations in which labelled carbon, nitrogen, sulphur, 
etc. are applied as indicators. 



EXTRACELLULAR AND INTRACELLULAR ORTHOPHOSPHATE 

It is probable (Sacks, 1949) that orthophosphate participates in 
phosphorylation processes during its passage through the cell bound- 
ary. It is likely that 32p reaches the cells as ATP P3 or ATP P23 
and then gets into rapid exchange equilibrium with the cellular ortho- 
phosphate. 

The importance of the intracellular orthophosphate P as a precursor 
—even if it were not the ultimate one— is enhanced by the 
fact that within a very short time intracellular orthophosphate P and 
ATP P3 (and to a large extent even ATP Pg) show the same activity 
level. This is of interest both in view of the fact that ATP is an im- 
portant phosphate donor and also because the specific activity of ATP P3 
is a more reliable measure of the specific activity of the genuine intra- 



TUBXOVKIl OF PHOSPHATIDES 349 

cellular orthophosphate than the value obtained in the direct deter- 
mination of tissue orthophosphate, even if the extraction was preceded 
by viviperfusion. 

In the case of the rat liver, which is showing both a high capillary- 
wall and a high cell membrane permeability, we found in all our experi- 
ments lasting 2 hours to 84 days within ±10 per cent identical values 
between the specific activities of tissue orthophosphate P and ATP Pg, 
P3, indicating that in these cases it is not advantageous to consider the 
specific activity of ATP P^, P3 instead of the corresponding value of 
the tissue orthophosphate P. In experiments, however, in which the 
formation of labelled phosphatides in the mouse liver was investigated, 
only 10 min following intravenous injection of labelled phosphate, 
a marked difference between the specific activity values of orthophos- 
phate P and ATP Pg 3 was found. Taking the specific activity of the 
plasma orthophosphate P to be 100, the corresponding values of liver 
orthophosphate P and ATP P23 were found to be 31.9 and 21.5, respect- 
ively. Assuming the extracellular volume (25%) of the liver to have 
the same concentration (5 mgm%) and specific activity as the plasma 
orthophosphate P, we find in 100 gm liver 1 .3 mgm extracellular orthophos- 
phate P having an activity of 1.3x100 = 130, while the total tissue 
orthophosphate P, the concentration of which is 32.3 mgm%, has an 
activity of 1030; thus, in this case, the activity of the extracellular orlho- 
])hosphate P makes out 13 % of the activity of the total liver ortho- 
phosphate. In experiments of such short duration it is advisable to con- 
sider the specific activity of ATP Pg 3 or preferably that of ATP P3, 
as a measure of the specific activity of the intracellular orthophosphate P. 

As another case in which the specific activity of ATP P, 3 has to be 
regarded as a measure of the specific activity of the intracellular ortho- 
phosphate P, that of the lung may be mentioned. One hour after admi- 
nistration of labelled phosphate by subcutaneous injection to 170 gm 
rats, the relative specific activity values (the plasma orthophosphate 
value being taken = 100) for the tissue inorganic P and ATP Pg 3 were 
found to be 52.7 and 35.6, respectively, the higher value of the specific 
activity of the tissue inorganic P being possibly due to the presence 
of highly active extracellular orthophosphate P in the tissue orthophos- 
phate investigated. 



CALCULATION OF THE RATE OF RENEWAL 

In all previous calculations, except a quite recent one to be discussed 
on p. 358, carried out hitherto it was assumed that labelled phosphate, 
or another precursor which comes into rapid exchange equilibrium 
(compared with the rate of formation of the lal)elled ])hosphalides) 



350 ADVENTURES IN RADIOISOTOPE RESEARCH 

with the labelled phosphate, is incorporated into the labelled phos- 
phatides. If this assumption, holds and 1 mgm inorganic intracellular 
liver phosphate contains throughout the experiment 1000 ^ap atoms, 
the presence of 10 ^ap atoms in 1 mgm of phosphatide P at the end of 
the experiment, lasting for example 1 hour, indicates that 1 per cent of 
the phosphatide molecules present in the liver is formed during the 
experiment. This figure indicates the percentage labelled molecules. As 
some of the molecules will be turned over twice or even several times 
during the experiments, and the formation of one labelled phosphatide 
molecule from another labelled phosphatide molecule is not registered 
by this method, the number of phosphatide molecules turned over in 
1 hour will be greater than 1 per cent. If the average concentration of 
the labelled molecules was 0.5 per cent during the experiment, about 1 
per cent of this 0.5 per cent would be ''invisibly" renewed a second 

time. 

The total percentage of phosphatide molecules turned over will 
thus be 1.005 per cent. The effect of more than 2 renewals of the same 
molecule in experiments of restricted duration can be disregarded 
and the same applies, in experiments of short duration, even for the 
second renewal of a molecule, as the percentage error of this type of 
experiments is quite appreciable. In the recent work by Bollman and 
Flock (1948), for example, in which remarkably uniform results were 
obtained, the deviations of the specific activity values obtained for both 
the inorganic P and the phosphatide P are still of the order of ± 10 

per cent. 

In view of this fact an exact evaluation of turnover rates is often 
without interest. However, if such an evaluation is required, one is 
tempted to take into account, beside the repeated renewal of the phos- 
phatide molecules in the course of the experiment, the constant dilution 
of the labelled molecules by non- (or sHghtly) labelled ones which pene- 
trate from the plasma into the liver. In the dog, 2 per cent of the liver 
phosphatides were found by Zilversmit et al. (1943 b) to be replaced 
by plasma phosphatides in the course of 1 hour, a somewhat greater 
replacement being found by Hevesy and Hahn (1940 b) in the liver 
of the rabbit. Assuming the liver of the rat to show a similar behaviour 
to that of the dog, about 0.1 per cent of the phosphatide content of the 
liver will be replaced by plasma phosphatide molecules in the 1 hour- 
experiment. 

In our considerations we assumed the specific activity (activity per 
mgmP)oftheorthophosphate P of the liver to remain constant during 
the experiment. This is not strictly correct. In former experiments 
(Ahlstrom and assoc, 1944) the mean value of the specific activity 
of the orthophosphate P of the rat liver during a 2 hour-experiment 
was found to be about 5 per cent larger than the end value. In 



TURNOVER OF PHOSPHATIDES 351 

recent experiments Ave found this figun^ to be aboiil 10 ])rv cent. Tn 
these experiments the lal)elled sodium ])h()sphate was a(hninis1ered l)v 
subcutaneous injection. If, however, the labelled phos])hate is given 
by intrav(>nous injection, as in some of the experiments of Bollman 
et al. (1948), a greater difference, viz. 24 per cent, is found between 
the mean and the end value of the specific activity of the orthophosphate 
P. While subcutaneous injection is foUowc^l by an increase in the speci- 
fic activity values of the plasma inorganic P during the first phase of the 
experiment followed by a comparatively slow decrease, intravenous 
injection is followed by a rapid decrease in the specific; activity of the 
plasma inorganic P which is the precursor of the liv(>r inorganic P. 
Intravenous injection is thus a less favourable procedure when we aim 
at a constant activity level of the orthophosphate P of the liver in 2 
hour-experiments. In experiments like the last mentioned one, and also 
when maximum precision is wanted, even in the first mentioned type 
of experiments we have to take into account the change of the specific 
activity of the orthophosphate P during the experiment. 

The percentage ratio of the specific activities of the end value of 
the phosphatide P and of the mean value of the orthophosphate P 
during the experiment indicates the lower limit of the percentage of 
new phosphatide molecules (new as to their P content) formed during 
the experiment. As already mentioned above, if we want tf) know the 
percentage of phosphatide molecules turned over during the experi- 
ment, we must consider among others a repeated renewal of the same 
molecule as well and the percentage ratio of the mean specific activities 
of the phosphatide P and the inorganic P during the experiment. In 
most experiments of restricted duration, for example in our experiments 
taking 2 hours, the correction due to the repeated renewal lies well 
within the errors of the experiment. 

In experiments of long duration in which a large percentage of the 
phosphatides is renewed, the calculation carried out by Zilveesmit 
et al. (1943 a) can be used with advantage. This calculation is based 
on the change of the specific activity of the phosphatide P with time; 
specific activity values at different times have thus to be known as well 
as the mean value of the specific activity of the inorganic P during 
the experiment. While the last mentioned method is generally appli- 
cable, the first mentioned method can only be applied advantageously 
when the percentage turned over is not over 20 — 30 per cent. Its advant- 
age is that the knowledge of one phosphatide activity value, that deter- 
mined at the end of the experiment, suffices to calculate the turnover 
rate. If a correction of the repeated renewal of the same molecule is 
wanted, it suffices to assume that the mean value of the specific 
activity of the phosphatide P during the experiment is half of its 
end value. 



^52 



ADVENTURES IX RADIOISOTOPE RESEARCH 



FORMER INVESTIGATIONS 

In the first investigations (Hevesy and Hahn, 1940) in which the 
specific activity of the plasma inorganic P of the rabbit was kept con- 
stant during an experiment lasting 4 hours, the percentage renewal 
of the phosphatides with incorporation of intracellular orthophosphate 
or an organic phosphate which comes into rapid exchange-equilibrium 
with the intracellular orthophosphate, was found to be 16. In the experi- 
ments by Hahn and Tyren (1946) the mean value of the specific acti- 
vity of the intracellular orthophosphate P of the liver was not determined. 
A comparison of the values obtained for rabbit and rat livers shows, 
however, that phosphatides are turned over at an appreciably more 
rapid rate in the liver of the rat. In experiments of 2 hours' duration, 
Hevesy (1947) found a percentage renewal with incorporation of intra- 
cellular orthophosphate of 7.6 per hour for phosphatides of the whole 
liver tissue of the rat and a value of 4.5 only for the percentage renewal 

Table 1. — Calculation of Bollman et al.'.s Experimental 
Data of the Turnover of Liver Phosphatides 





Duration of experiment 
in liours 


Percentage renewal per lir 




Calculated 

according to 

ZILVERSMIT 

et al. 


Calculated according to HEVESY 
and Hahn 




Without consid- 
ering repeated 
renewal 


Considering 
repeated 
renewal 


5 




4.95 
5.17 
5.34 
4.93 


4.89 
4.04 
6.08 
4.39 


4.95 


1 




5.16 


9 




5.34 


4 




4.931 









(^> If, instead of considering tlie Ivnown mean phospliatide specific activity during tlie experiment, we 
■consider V2 of tlie end value, 4.83 is obtained. 



of the phosphatides present in the cell nuclei. Bollman et al. 
(1948) arrived at a figure of 5.3 for the percentage renewal of the liver 
phosphatides in the course of 2 hours. These authors raise doubts as 
to the applicability to their case of the method of calculating the turn- 
over rate as outlined above (Hevesy and Hahn, 1940 a) and perform 
their calculations by applying the method worked out by Zilversmit 
ei al. (1943). That the discrepancy between the percentage turnover 
of the liver phosphatides of the rat as found by Bollman et al. 
and by the present authors is not due to a difference in the method 
of calculating the experimental figures obtained, is demonstrated in 
Table 1, in which the results of the evaluation of the experimental data 



TURNOVER OF PHOSPHATIDES 353 

of BoLLMAN et aJ. are calculated both according to their and our 
method, almost identical figures being obtained in l)oth cases. 

The method of calculating the experimental results can thus not 
be responsible for the difference in the renewal percentage obtained 
by different workers and we have thus to consider other explanations. 

As shown in the present communication, the ratio of the specific 
activities of the orthophosphate P and phosphatide P of the liver varies 
with the age of the rat, and these variations can be assumed to be at 
least partly responsible for the discrepancies mentioned al)Ove<^\ 

EXPERIMENTAL 

To each of more than 100 rats of known age, kept on normal diet, 0.1 ml physiol. 
NaCl solution containing 32p of 2 /(curie activity and a negligible ^ip content 
was administered by subcutaneous injection. Two hours later, batches of 4 or 
irioro rats were pooled, the animals were killed by decapitation, bled, and the 
isolated organs frozen with sohd COg. An aliquot of the liver, spleen and kidney 
samples was used to determine the specific activity of the inorganic P, another 
for the total P, while a third was used for the determination of the specific activity 
of phosphatide P. In the case of the Uver, the labile P of ATP was investigated 
as well. All organs were cut into small pieces and average fractions were obtained. 
To measure the inorganic P values, fractions (0.2 — 0.3 gm) were extracted with 
cold CCI3COOH. The total P was obtained by wet ashing of about 0.2 gm fresh 
tissue, while a few gm were used for the extraction of phosphatides. 

Before extracting phosphatides the tissue was treated with 200 ml acetone 
for 15 minutes. The filtrate was dried in a COg atmosphere and the residue extracted 
with ether. The acetone treated tissue was extracted by grinding it in a mortar 
twice with 150 ml ether and once with 1 : 3 ether-alcohol mixture for 15 minutes. 
The residue was then extracted for 8 hours in a Soxhlet flask with 150 ml boiling 
ether-alcohol mixture (1 :3). All ether and alcohol fractions were united and diied 
in a CO2 atmosphere. To free the phosphatides from traces of inorganic P and 
other P compounds the residue was dissolved in 300 ml ether and shaken with 
450 ml 0.1 n HCl -|- 0.01 n NaCl solution in a separating funnel. This procedure 
was repeated four times, as suggested by Hahn and Tyren (1945). 

The ethereal solution was evaporated in a Kjeldahl flask and then ashed by 
a mixture of HgSO. and HNO3. An idiquot of this solution was used for colorimet- 
ric determination and another precipitated as magnesium ammonium phosphate 
and its radioactivity determined. 

To determine the specific activity of the labile P of ATP about 8 gm of liver 
tissue were extracted with 3 volumes cold CCI3COOH solution. The cooled filtrate 
was neutrahzed to phenolphtalein with cooling by adding solid Ba(0II)2. 

The precipitate containing adenosine triphosphate, adenosine diphosphate, 
orthophosphate, and some other minor fractions of organic P compounds was 
washed with a little Ba(0n)2 and neutralized with CClgCOOII. Subsequently 
it was dissolved in 15 ml n HNO3. To the solution, as suggested by Sacks and 
Altschuler (1942), NH^NOg was added until a concentration of 5 per cent was 

(1) That the spec, activity of the liver P of mice declines from 3.7 to l.G when 
the age increases from H to 24 weeks was observed by Falkenheim (1943). 

23 Jievesy 



354 



ADVENTURES IN RADIOISOTOPE RESEARCH 



obtained, followed by 2 ml 10 per cent ammonium molybdate solution. The inorga- 
nic P was precipitated overnight; the filtrate was then hydrolyzed for 20 minutes 
at 100°C and cooled. The precipitate contained the labile P of ATP. This was 
dissolved in 15 ml 5 per cent NHg and its P precipitated as magnesium ammonium 
salt. The precipitate was dissolved in 0.1 n HCl, an aliquot being used in the 
eolorimetric essay, while another was precipitated as magnesium ammonium salt 
and reserved for the radioactive measurements. 

Percentage Turnover of Liver Phosphatides 

The lower limit of the percentage turnover of liver phosphatides 
per hour, which is calculated from the percentage ratio of the speci- 
fic activities of the liver phosphatide P at the end of the 2 hour-experi- 
ment and the mean value of the orthophosphate P during the experi- 
ment (which was by 10 per cent less than the end value) and divided 
by 2, is given in column 2 of Table 2, while column 3 contains values 



Table 2. 



Lower Limit of the Percentage Turnover per 



HotTR OF THE Phosphatides of the Rat Liver. ATP Pj 3 Scale 



Age of rats 



Percentage turnover per liour 



AVithout considering 
repeated renewal 



Considering 
repeated renewal 



4 d 

10 d 

14 d 

30 d 

10 d 

1.5 year(i) 

<'' Calculated from inorganic P value. 



12.1 ± 1.2 

9.9 ± 1.0 

10.0 ± 1.0 

9.6 ± 1.0 

7.7 ±0.8 
5.7 + 0.6 



12.7 ± 1.3 

10.4 ± LI 

10.5 ± l.l 
10.1 ± 1.0 

8.1 +0.8 
6.0 + 0.6 



corrected for the repeated renewal of phosphatide molecules during the 
experiment. The correction is obtained by calculating the percentage 
ratio of the average specific activities of the phosphatide P and the 
orthophosphate P during the experiment. The average ratio of the spe- 
cific activity of the phosphatide P was taken to be half of the end value 
(cf. footnote, p. 353). The turnover rate is seen to decrease with the age 
of the rats, the percentage turnover rate of the phosphatides of 1.5 
year-old rats being only about half of that obeserved in 4 day-old rats. 

In Table 3 the percentage turnover calculated by comparing the speci- 
fic activity of the phosphatide P with that of the inorganic P is shown. 

BoLLMAN et al. (1948) found the percentage turnover per hour to 
be about 5. From the fact that their rats weighed 200 gm we have to 
conclude that the animals investigated were fully grown rats for which 
we arrive at a corresponding figure of 8 to 6. Furthermore, we have to 



TURNOVER OF PHOSPHATIDES 



355 



consider that in contrast to the present autliors, lliey investigated lasting 
rats. Platt and Porter (1947), when comparing the turnover of plios- 
phatides in the liver of fed and fasting rats, found the former value to 
be about 1/3 larger than the latter. That the diet influences the percentage 
renewal of liver phosphatides of the rat was also shown recently by 
Campbell and Kosterlitz (1948). 

While we cannot calculate turnover rates from 1h(> ratio of the speci- 
fic activities of the phosphatide P and total liver P, this ratio indicates 
to what extent phosphatide P atoms are renewed compared with the 
average total P atoms. We listed these ratios in Tabk; 4 along witli the 
total P content of the livers investigated. We have not listed the inor- 
ganic P nor the phosphatide P contents as we were interested primarily 
in phosphatide P and inorganic P fractions of high purity and the ex- 
tended purification processes entailed an appreciable loss. 

The absolute amount of phosphatides renewed during a given span 
of time increases, in contrast to the percentage renewal, with the age 
of the rat, as both the weight of the liver and its phosphatide content 
are increasing with age. The phosphatide content of the liver of the 

Table 3. — Lower Limit of the Percentage Turnover per 
Hour of the Phosphatides of the Rat Liver. Inorganic 

P Scale 





Pei-ceutage turnover per hour 


Age of rats 


Without considering 
repeated renewal 


Considering 
repeated renewal 


4 d 

10 d 

14 d 

90 d 

1.5 V 


10.2 ± 1.0 

9.8 ± 1.0 
10.4 ± 1.0 

7.9 ±0.8 
5.7 ±0.6 


10.7 ± 1.1 

10.3 ±1.0 

10.9 ± 1.1 

8.3 ±0.8 

6.0 ±0.6 





Table 4. — Specific Activity of Liver Phosphatides as 
Percentage of the Specific Activity of the Total P of 

the Liver 



Age of rats 


Percentage specific 
activity 


Xutal P 


of liver in mgin 

P.O. 


4 d .. 




67 




324 


10 d . . 




64 




318 


14 d .. 




74 




363 


30 d . . 




63 




348 


90 d . . 




43 




374 


1.5 y 




58 




370 



23* 



356 



ADVEXTURES IN RADIOISOTOPE RESEARCH 



90- day-old rat (3200 mgm%) is % times that of the 4-day-old rat (2800 
mgm%) and the weight of the liver increases simultaneously from 0.24 gm 
to 4.0 gm (Lang 1937). The phosphatide content of the 90-day-old rat 
is thus 20 times that of the 4-day-old rat. The percentage turnover of 
the 4-day-old rats being 1.5 times that of the 90-clay-old animals, the 
amount turned over in the course of 1 hour in the 90-day-old animals is 



lO 
































































+ 






























































in 


° i 


o 


. 


. 










































































( 


) 
































&0 












































5 
















































































































+■ InorganicPscaie 
O ATP, P^^ scale 






















n 

























540 



10 20 30 40 50 60 70 80 90 

Age in days 



540 



Fig. 1. Percentage incorporation of orthophosphate P resp. ATP P^.z 
per hour into liver phosphatides of rats of different age 

15 times that renewed in the 4-day-old rats, the figures being 10.25 mgm- 
and 0.69 mgm, respectively. If between the phosphatide and the ortho- 
phosphate (or ATP) molecule a phosphorus compound were interposed 
which was formed at a comparatively slow rate, it would be the ultimate 
precursor of the phosphatide molecule ; then the above mentioned figure 
would represent the lower limit of the amount of phosphatides turned 
over only. 



Spleen Phosphatides 

Since we do not know the mean value of the orthophosphate P of 
the spleen during the experiment we cannot calculate the lower limit 
of turnover rate. Assuming the mean value to be 2/3 of the end vahic 



TURNOVER OF PHOSPHATIDES 



35" 



for the 90-day-okl rat, we arrive at a lower limit of percentage turnover 
of 3 per hour by making use of the data given in Table 5. This is a rough 
estimate which indicates that the turnover rat(> is about 1/3 of that found 
for the liver phosphatides. 

Table 5 contains data of the relative specific activities of the spleen 
total P and phosphatide P and the plasma inorganic P, 1he spleen 
inorganic P value being taken to be = 100. 



T.'VBLK 5. — Specific Activity of Spleen Phosphatidks 
Experiment Taking 2 Hours 



Age of 


nxts in da.YS 


rerceiitagc ratio of the specifiuactivity of iiliosphatido 
I' to tlKlt of 


Spleen inorgsmic 
P 


Spleen total 
P 


Plasma inorganic 
P 


4 

10 


4.8 
5.2 
5.0 
3.9 
3.8 
3.4 


13.7 
17.0 
18.3 
18.5 
13.8 
16.0 


4.9 

4.8 


14 




30 

«J0 


3.4 
2.3 


540 


1.6 


Table 


6. — Specific Activity of Kidney Phosphatides 
Experiment Lasting 2 Hours 


Age oi: 


I'iits iu (lays 


Percentage ratio of the spec, activity of phosphatide 
P to that of 


Kidney inorganic 
P 


Kidney 
total P 


Plasma inorganic 
P 


10 

14 

30 

90 

540 


14.0 
14.4 
15.2 
12.1 
12.1 


44.0 
37.2 
41.3 

27.9 
37.8 


10.3 
10.6 
13.5 
11.2 
11.6 



The percentage ratio of the specific activities of the phosphatide P 
and spleen inorganic P measured at the end of the experiment decreases 
with the age of the rat, the decrease being more pronounced if we con- 
sider the ratio of the percentage activities of the phosphatide P and 
plasma inorganic P, as seen in Table 5. This decrease indicated a decreas- 
ing permeability with age of the spleen cells to inorganic P, a fact 
previously observed by Ahlstrom et al. (1944) and by Andre asen 
and Ottesen (1945). 



358 



ADVENTURES IN RADIOISOTOPE RESEARCH 



Kidney Phosphatides 

The ratio of the specific activities of kidney phosphatide P and inor- 
ganic P is appreciably higher than the corresponding spleen values, 
though it falls below the liver values. We found the mean value of the 
specific activity of the kidney inorganic P to be 1.35 times the end value 
and, consequently, we have to divide the figures of column 3 of Table 6, 
which denote data of experiments, taking 2 hours, by 2x1.35 in order 
to arrive at an estimate of the lower limit of the percentage turnover 
per hour. 

The pronounced decrease in the phosphatide turnover with age shown 
by the liver phosphatides is not exhibited by the kidney phosphatides. 

Turnover of Lecithin and Cephahn 

In the investigations described above the rate of incorporation of 
22P into the total liver phosphatides was determined. Lecithin and 
cephalin are renewed at a not very different rate. Platt and Porter 
(1947) found, for example, 6 hours after administration of labelled 
phosphate to full-grown rats kept in usual diet the ratio of specific 
activities of lecithin P and cephalin P to be 1.5, while in the fasting rat 
1.3 was found. These authors state, furthermore, that administration 
of choline increases the turnover rate of lecithin while ethanolamine 
promotes the turnover of cephalin formation. In the first mentioned 
case a maximum increase of 33 per cent, in the latter case a maximum 
increase of 87 per cent of the turnover rate was observed. They inter- 
pret the increased formation of labelled phosphatides following admi- 
nistration of choline and ethanolamine, respectively, as a mass action, 
the assembly of the phosphatide molecule being promoted by an increase 
in the choline and ethanolamine concentration, respectively. 

In experiments on dogs, Zilversmit and assoc. (1948) recently found 
the mean specific activity ratio of lecithin P and cephalin P to be 1.2. 

Table 7. — Specific Activities 



Fraction 


Specific activity' 


Plasma orthophosphate P 

Liver orthophosphate P 

Glycei'ophosphate P 

Total P 


0.546 
0..550 
0.376 
0.180 


Total ti.ssue phosphatide P 

Mitochondria phosphatide P 

Cell nuclei phosphatide P 


0.0705 
0.0473 
0.0296 



' Activity of 1 mgm I' in percentage of tlie activity administered to tlie rat. 



TURXOVEll OF PHOSPHATIDES 



359 



Phosphatide Turnover in Cell Nuclei and Mitochondria 

The figures obtained by difi'erent investigators for ilic turnover 
rate of phosphatides (or for the turnover rate of lecithin, cephalin) 
are average figures, as the turnover rate in different types of cells and 
even in different parts of the same cell (cf. Hevesy, 1947) differ. In 
Tables 7 and 8 the specific activities of the various isolated fractions 
resp. the percentage replacement of phosphatide P of the total liver 
tissue of the cell nuclei and of the mitochondria of Ihe liver by tissues 
inorganic P, glycerophosphate P and total liver, resp., P is stated. 
Isolating glycerophosphate we made use of the method applied by 
Entenman et al. (1948). 6 ^curie of ^sp were administered by subcuta- 
neous injection to each of 6 rats weighing about 150 gm. The animals 
were killed after the lapse of 2 hours. 

The phosphatide P turnover of mitochondria makes out 67% only 
of the corresponding figure of the total phosphatide P while the corre- 
sponding figure for the cell nuclei is 42 only. The last mentioned figure 
was in a former investigation found to be 65. The discrepancy is clue 
to the fact that in the method used formerly of separation of cell nuclei 
(DouNCE, 1945) the nuclei containing fraction contained also mito- 
chondria, in which as seen above, phosphatides are turned over at a 
more rapid rate than in the cell nuclei. 

The activity of 1 mgm phosphatide P in percentage of the activity 
of 1 mgm liver orthophosphate P listed in Table 8 indicates the lower 
limit of the percentage replacement of phosphatide P in the course of 
the experiment taking 2 hours. By a fortuitous coincidence, the end 
value and the average value, of the specific activity of liver orthophos- 



Table 8. — Percent.4igk Replacement of the Phosphatide P of 

THE LrV'ER 



Phosphatide fr;»L'tion 


Activit)- of 1 mgm phosphatide P in percentage of the activity 
of 1 mgm P of 


Plasma 
orthophosphate 


liver 
ortophosphate 


liver glycero- 
phosphate 


liver total P 


Total tissue 

Mitochondria 

Cell nuclei 


13.4 
9.37 

5.64 


12.8 
8.95 
5.38 


18.75 
13.1 

7.89 


39.2 
27.5 
16.5 



phate are almost identical in an experiment taking 2 hours. This is not 
the case for the specific activity of gylcerophosphate P, as the labelled 
glycerophosphate accumulates gradually in the course of the experiment 
by incorporation of labelled orthophosphate P. We can assume the 
average value of the specific activity of glycerophosphate P during the 



360 



ADVENTURES IN RADIOISOTOPE RESEARCH 



experiment to be about half its end value. Correspondingly, we have 
to multiply the figure 18.75 listed in Table 8 by 2 to arrive at the per- 
centage renewal of the liver phosphatides, assuming glycerophosphate 
to be the last phosphatide precursor; when, on the other hand, assuming 
orthophosphate P to be a relevant precursor, a value of 12.8% is obtained. 
The turnover rate calculated on glycerophosphate "basis" is thus about 
3 times the value obtained, supposing that orthophosphate P is the 
relevant precursor. 

In experiments on dogs, Zilversmit et al. (1948) found a similar 
ratio (3.5) for the turnover rate of liver lecithin calculated on the assump- 
tion that glycerophosphate P resp. orthophosphate P is the ultimate 
phosphatide P precursor. Assuming glycerophosphate to be the pre- 
cursor of phosphatides, the turnover time of the liver phosphatides 
of the 150 gm rat works out to be 5 hours. 

As stated on p. 359, several arguments were put forward in support 
of the view that glycerophosphate is the ultimate precursor of phospha- 
tides and that the comparison of the specific activity of the liver phos- 
phatide P with the corresponding value of the liver glycerophosphate 
P supplies a 3 times as high, and correct, turnover rate, as does compari- 
son with the liver orthophosphate P. This would indicate that it is the 
formation of labelled glycerophosphate which takes comparatively 
k>ng time, while the incorporation of labelled glycerophosphate into 

Table 9. — Effect of Feeding of Labelled Glycerophos- 
phate RESP. Labelled Orthophosphate on the Formation of 
Labelled Phosphatides in the Liver of the Rat 

(Artom and Swanson 1948) 



Specific activity (arbitrary units) 


Labelled glycero- 
phosphate fed 


Labelled ortho- 
phosphate fed 


Liver glycerophosphate P . . . . 

Liver orthophosphate P 

Liver phosphatide P 

Plasma phosphatide P 


45.3 
21.6 

8.1 
0.5 


18.0 
23.4 

6.9 
6.8 



the phosphatide molecules is performed at a comparatively rapid rate. 
This conclusion is not easy to reconcile with a recent finding by Artom 
and Swanson (1948). These authors state that 6 hours following feeding 
of labelled glycerophosphate, the specific activity of liver glycerophos- 
phate P was much greater than the corresponding value of orthophos- 
phate P of the liver and also much greater than the orthophosphate P 
value of the liver following feeding of labelled sodium phosphate. The 
specific activity of phosphatide P of the liver was, however, not signi- 
ficantly greater in these experiments in which a high liver glycero- 



TURNOVER OF PHOSPHATIDES 



361 



phosphate specific activity was observed. The presence of a highh^ 
active glycerophosphate in the liver did thus not markedly accelerate 
the formation of labelled phosphatides. The above stated specific acti- 
vity figures were found by Artom and Swanson 6 hours aflci' feeding 
of the labelled compounds. 

In view of the fact that the results of only one experiment is stated, 
the results of this investigation, which aimed at the elucidation of a 
very different problem, can however not be considered to invalidate 
ZiLVERSMiT et al. conclusions. A closer investigation of the phos- 
phatide formation in the liver following feeding of labelled glycero- 
phosphate would not be without interest. 



Search for the Existence of a Small Phosphatide Fraction of Rapid 

Turnover Rate 

It is conceivable that a small fraction of phosphatides is present in the liver, 
which is renewed at a more rapid rate than the average phosphatide molecule 
present. The existence of such a fraction could influence much the conclusions 
drawn in the last paragraph which are based on the number of phosphatide mole- 
cules turned over during a time unit. 



Table 10. — Specific Activities of P Fractions of Mice 10 

MlNT'TES FoLLOWaXG InTRAVENOT'S InJECTIOX OF ^"P 



Fraction 


Specific activity' 


1 2 


3 


Plasma orthophosphate P 

Liver orthophosphate P 

Liver ATP P„ , 


20.4 
6.53 
4.30 
0.0983 


32.6 

4.26 
0.0898 


31.8 
6.56 


Liver phosphatide P 


0.0813 



' Activity of 1 mgm P in percentage of the activity administered. 

Let us assume that of 100 phosphatide molecules 99 are renewed to an extent 
of 18% in 1 hour, while 1 phosphatide molecule is renewed 100 times during the 
experiment. Our experiments would hardly reveal the presence of such a fraction 
responsible for 118 renewal processes, while our turnover experiments would 
reveal a 19% renewal of the average phosphatide molecules, only. One hundred 
renewals pei- hour is a large figuie in view of the fact that the very i-apidly rejuv- 
enated ATP Pg 3 in experiments in vitro is found to be renewed 72 times per 
hour only (Meyerhof et al., 1938). But even a 1% phosphatide fraction renewed 
10 times in the course of 1 hour would be responsible for 10 renewal processes 
beside the 18 performed by the rest of 99 "^o phosphatide molecules, increasing 
the number of molecules turned over to 28. 

The presence of a small rapidly renewed fraction should become noticeable^ 
in experiments of very short duration. 

We carried out experiments in which about y^ microcurie ^zp was injected 

into the tail vein of each group of 10 mice; the (22 — 26 gm) animals were killed 
by decapitation idler the lapse of 10 minutes, the organs pooled, and the specific 



362 



ADVENTURES IN RADIOISOTOPE RESEARCH 



activity of the plasma ortliophosphate, liver inorganic P, liver ATP Pg^g and 
phosphatide P determined. Some of the resuhs obtained are listed in Table 10. 
The phosphatides were purified according to Levin's method as modified by 
Hahn and Tyren (1946). The above results do not indicate the presence of a 
rapidly renewed phosphatide fraction. 



Experiments of Long Duration 

As observed at an early date (Hevesy and Aten, 1938: Zilversmit et al., 
1943, 1948) in experiments of long duration the specific activity of phosphatide 
P exceeds the corresponding value of orthophosphate P, the last mentioned magni- 
tude decreasing at a slower rate than the orthophosphate P. As seen in Table 11 
the specific activity of orthophosphate P resp. labile ATP phosphorus declines 
between 1/12 day and 84 days (the maximum value is observed about V12 day after 
subcutaneous injection) from 0.546 to 0.000603. Thus, out of 900 ^ap atoms in the 

Table 11. — Specific Activity of Liver Fractions at Different Dates Following 

Administration of ^-P 



i 






Specific activity' after 






Fraction 


1/12 day 


1 day 


12 days 


21 days 


44 days 


84 days 


Orthophosphate P . . . 
ATP P„ „ 


0.546 
0.00703 


0.108 
0.105 
0.121 


0.0153 

0.0185 
0.0252 


0.00849 
0.00481 
0.00518 


0.00134 

0.00169 


0.000603 


^-^■^ ^2,3 

Phosphatide P 


0.00066 



1 Activity of 1 mg P in percentage of the dose administered, obtained by comparing tlie radioactivity 
oi a known aliquot of the solution injected with that of a known amount of phosphorus of the fraction 
mvestigated. 

nitial maximum state, only 1 is present after the lapse of 84 days (beside the loss 
due to radioactive decay). During the same interval, the beginning of which 
does not correspond, however, to the maximum value of ^ap content of phosphati- 
des, the 32P content of phosphatides decreases to Viio of their initial value. 

The decline of the ^sp of liver orthophosphate, which in view of the highperme- 
abihty of liver cells corresponds closely to the fall of ^ap content of plasma 
orthophosphate, is due partly to incorporation of ^ap into the tissues and partly 
to its excretion. With increasing time excretion becomes more and more the sole 
way of escape of ^sp from the circulation. While on the first day, the ^ap content 
of orthophosphate decreases to V5 oi its 2-hour-value, a decline to V7 of the first 
day's value takes 12 days, a decline to V3 of the 12 day- value 9 days, the decrease 
of the 21st day value to almost 1/4 takes not less than 28 days, while a decrease 
of half the 44 day- value requires 35 days. 

The renewal of the phosphatides takes place at a slower rate than the escape 
of 32p from the plasma, however, after a long sequence of days this difference 
diminishes due to the reduced loss of ^ap by the circulation. 

The values were obtained from pooled organs of 4—6 rats weighing about 
160 gm. In the 84-day experiment 0.1 miUicurie was administered to each rat, 
in the experiments of shorter duration correspondingly less. The administration 
of 0.1 millicurie or more involves risks in experiments of long duration as to bio- 
logical action of the radiation emitted by the administered P, as the radiation 
dose to which the rats are exposed during the experiment may amount to a few 
hundred rep. 



TUENOVKll OF PHOSPHATIDES 363 

Fat Metabolism and Phosphatide Turnover 

A possible connection between fat metabolism and phosphatide turn- 
over was repeatedly discussed. Bollmam and Flock (1946) compared 
the amount of phosphatides turned over in the liver and plasma with 
the amount of fat metabolized by the rat and arrived at the following 
conclusion. 

Assuming, as found by these authors, 0.175 mgm of phosphatide P 
to be renewed per hour in a rat liver weighing 5 gm, an equal amount 
of phosphatide P must have been metabolized in the liver or have 
left this organ during that time, the latter amounting to 0.048 mgrii. 
These figures account for a sufficient phosphatide turnover in the liver 
and in the plasma to metabolize or transfer fat equivalent to only 3 per 
cent of the caloric needs of the rat and indicate that phosphatide forma- 
tion apparently is not an obligatory step in fat oxidation or transfer. 

For the turnover rate of the 150 gm rat we found about twice the value 
given by the above authors. Thus, according to our results, the amount 
of phosphatides turned ovei- should be twice the above figure and, 
if glycerophosphate is assumed to be the ultimate phosphatide pre- 
cursor, even three times the last mentioned value, but still only 18 per 
cent of the caloric needs of the rat. Consequently, our findings do not 
contradict Bollman and Flock's conclusion. 

In the above consideration no account was taken of the possibility 
of the existence of a minor phosphatide fraction which may be renewed 
very rapidly, as discussed on p. 361, nor was the phosphatide turnover 
in other organs than the liver accounted for. 

Summary 

(1) The extent of incorporation of intracellular ortliophosphate P of the liver 
phosphatides of the rat decreases with increasing age. While it amounts to 12 per 
cent per hour for the liver phosphatides of a 4-day-old rat, the corresponding 
figure for a 1.5-year-old rat is 6, intermediate figures being obtained for rats of 
intermediate age. 

(2) The calculation of the "rate of turnover" from the ratio of the end value 
of the specific activities of phosphatide P and the mean value of the specific 
activities of orthophosphate P during the experiment supplies, in experiments 
lasting 4 hours or less, almost identical figures with those obtained when, accord- 
ing to ZiLVERSMiT et al., the calculation is based on the change of the specific 
activity of phosphatide P with time. 

(3) Replacement of the specific activities of liver inorganic P by corresponduig 
values of ATP Pg^ of the liver leads to very similar turnover rate values. 

(4) The ratio of the turnover rate of the phosphatides of the total liver tissue, 
the mitochondria, and the cell nuclei of the liver is found to be 1 : 0.67 : 0.42. 

(5) The percentage of labelled P administered present in I mgm liver phospha- 
tide P of the rat, which is as high as 0.121 after the lapse of 1 day, declines to 
0.00066 after the lapse of 84 days. 



364 ADVENTURES IN RADIOISOTOPE RESEARCH 

(6) The lower limit of the turnover of kidney phosphatides is not markedly 
dependent on the age of the rat and amounts to about 5 per cent in the course 
of 1 hour. Lower and with the age of the rat decreasing values are obtained for the 
spleen phosphatides. The percentage rate of incorporation of orthophosphate P 
per hour for a three-month-old rat can be estimated to be about 4. 



References 

L. Ahlstbom, H. Euleb and G. Hevesy (1944) Ark. Kemi, A 21, No. 11. 

E. Andbeasen and J. Ottesen (1945) Acta Physiol. Scand. 10, 25. 

C. Abtom Arch. int. Physiol. 36, 101. 

C. Abtom, G. Sabzana, C Pebrier, M. Santangello and E. Segbe (1937) Nature 

139, 836. 
C. Abtom, G. Sabzana and E. Segre (1938) Arch. int. Physiol. 47, 245. 
C. Artom and N. A. Swanson (1948) J. Biol. Chem. 175, 871. 
J. L. BoLLMAN and E. V. Flock (1946) J. Lab. Clin. Med. 31, 478. 
J. L. BoLLMAN, E. V. Flock and J. Berkson (1948) Proc. Soc Exp. Biol. N. Y . 

67, 308. 
R. M. Campbell and H. W. Kosterlitz (1948) J. Biol. Chem. 175, 989. 
I. L. Chaikoff and D. B. Zilversmit (1948) Adv. Biol, and Med. Physics 2, 322. 
M. Falkenheim (1943) Amer. J. Physiol. 138, 175. 
L. Hahn and G. Hevesy (1937) Skand. Arch. Physiol. 77, 148. 
L. Hahn and R. Tyren (1946) Sv. Vet. Akad. Ark. Kemi, A 21, No. 11. 
G. Hevesy and L. Hahn (1940) Kgl. Danske Videnskab. Selsk. Biol. Medd. 

15 Nr. 5. 
G. Hevesy and L. Hahn (1946 b) Kgl. Danske Videnskab. Selsk. Biol. Medd. 

15, Nr. 6. 
G. He\-esy (1947) Sv. Vet. Akad. Ark. Kemi, A. 24, No. 26 
G. Hevesy (1948) Radioactive Indicators, Interscience Publ., New York. 
G. IvANOVics and E. P. Pick (1910) Wien. Klin. Wschr. 23, 573. 
A. Lang (1937) Z. Physiol. Chem. 246, 219. 

I. Perlman, S. Ruben and I. L. Chaikoff (1937) J. Biol. Chem. 122, 169. 
A. P. Platt and R. R. Porter (1947) Nature 160, 905. 
I. Sacks (1949) Cold Spring Harbour Symphosia 13. 
I. Sacks and E. H. Altschuler (1942) Amer. J. Physiol. 137, 1750. 
R. G. Sinclair (1936) J. Biol. Chem. 114. 94. 
R. G. Sinclair (1941) Biolog. Symp. 5, 82. 
D. B. Zilversmit, C. Entenman and M. C Fishler (1943) J. Oen. Physiol. 26, 

325; Ibid. 26, 333. 
D. B. Zilversmit, C. Entenman and I. L. Chaikoff (1948) J. Biol. Chem. 

176, 193. 



365 



Comment on papers 34—35 

We calculated in paper 29 and the following papers the rate of renewal of phospha- 
tides from the specific activity of the phosphatide phosphorus at the end of the 
oxperiment, and of the moan specific activity of the cellular inorganic phosphorus 
during the experiment. The latter is obtained from the specific activities of the 
total inorganic P after correcting for the share of the extracellular phosphorus 
in tlie total inorganic P activity. The possibility was considered in these studies 
that it is not the cellular but the extracellular ^^p which participates in the synthe- 
sis of the organic phosphorus compounds present in the tissues and that "It is 
conceivable that some of the phosphatide molecules are renewed inside the cell 
wall." Correspondingly, all turnover data were calculated, assuming once parti- 
cipation of cellular 32 p and than of extracellular ^sp (paper 34). That phospliate 
enters the cells, at least partly, by the formation of ATP and other intermediates 
of the cycle on the cell membrane and that inorganic phosphate within the cell 
arose from the dephosphorylating reactions of the cycle was shown later by 
Sacks (1951). 

In paper 36 in which the calculation of the turnover rate is discussed we find 
the following remark: " . . . If the incorporation of the phosphate radical into 
the phosphatide molecules would be preceded by the formation of glycerophos- 
phate and this process would be a comparatively slow one, in contrast to all other 
steps involved in the synthesis of the phosphatide molecule, in this case the turnover 
rate measured, using labelled phosphorus as an indicator, would be slower than 
fotmd when using labelled fatty acids or labelled choline . . ." and that "the 
question if and to what extent the rate of labelled phosphate incorporation into 
the phosphatide differs, for example fiom that of the fatty acid incorporation 
into the latter cannot be answered at the time being". The question thus raised 
was answered by Chaikoff, Zilversmit, and their associates (1941) who demon- 
strated that glycerophosphate is the pertinent precursor of phosphatide s,\Tithesis 
and that the calculation of the turnover rate of phosphatides from the specific 
activity of inorganic phosphorus leads to too low a value for the turnover rate. 
In experiments with rats, taking 2 hi', the mean specific activity of the ortho- 
phosphate phosphorus of the Uver was found to be three times that of the corre- 
ponding value of glycerophosphate phosphorus (paper 35); thus, the turnover 
rate calculated assuming the liver orthophosphate P to be the phosphatide pre- 
cursor has to be multiplied by three to arrive at a correct turnover rate value. 
The turnover rate of phosphatides present in the different sub-units of the liver 
cell was found to differ markedly (paper 35 and Hevesy, 1947). Applying palmitic 
acid — 1 — i^C as a precursor Chaikoff et al. could also demonstrate that the liver 
is the principal site for the formation of fatty acid ester bonds of plasma phos- 
phatide molecules. 

References 

O. Hevesy (1947) Ark. Kemi, A 24, No. 2C. 

J. Sacks (1951) Arch. Biochem. 30, 423. 

D. B. Zilversmit, C. Entenman, M. C. Fishler and I. L. Chaikoff (1941) 

J. Gen. Physiol. 26, 325. 
D. S. Goldman, I. L. Chaikoff, W. O. Reinhardt, C. En^tenman and W. G. 

Datjben, (1950) ./. Biol. Chem. 184, 727. 



Originally communicated in Nature 141, 1097 (1938) 



36. MOLECULAR REJUVENATION OF MUSCLE TISSUE 

G. Hevesy and 0. Rebbe 
From the Institute of Theoretical Physics and the Zoophysiological Laboratory 

University of Copenhagen 

The decomposition of creatine phosphoric acid during muscular action 
and its rebuilding during rest, has been the subject of numerous detailed 
investigations. We were interested in the problem, if, and to what 
extent, creatine phosphoric acid molecules are decomposed and after- 
wards rebuilt, or 'rejuvenated', in the resting muscle. This problem can 
be easily solved by injecting labelled sodium phosphate, for example, 
into frogs, and determining if, and to what extent, creatine phosphoric 
acid extracted from the muscle of the frog becomes labelled (radioactive). 
Phosphorus atoms present in creatine phosphoric acid and other organic 
compounds do not exchange spontaneously with other phosphorus 
atoms present, and thus the fact that labelled creatine phosphoric 
acid can be isolated from the muscle is a proof that this was synthesized 
after the administration of labelled sodium phosphate. 

The muscle was placed at once after removal in liquid air, the acid 
soluble components extracted with trichloracetic acid kept at —9°, 
and the inorganic phosphate present in the solution precipitated as 
ammonium magnesium salt. The next step was the decomposition 
of creatine phosphoric acid remaining in the filtrate from the last- 
mentioned precipitate. The decomposition was carried out by adding(^> 
sulphuric acid (1??) and ammonium molybdate (1 per cent) to the solution. 
The phosphate ions were then precipitated as ammonium magnesium 
salt. The phosphorus content of the latter was determined by the colori- 
metric method of Fiske and Subbarow, and its radioactivity by making 
use of a Geiger counter. The results obtained for this and some other 
fractions are seen in the accompanying table. 

A specific activity of the creatine phosphoric acid phosphorus amount- 
ing to 49 per cent of that of the inorganic phosphorus indicates that 
49 per cent of the creatine phosphoric acid molecules present in the 
resting muscle were split and newly synthesized through enzymatic 
action in the course of the last 3 hours before the frog was killed. As the 
total number of creatine phosphoric acid molecules present in the muscle 
can be assumed not to have changed during that time, in the resting 



MOLECULAR REJUVEXATIOX OF MUSCLE TISSUE 



367 



muscle we are faced with a molecular rejuvenation of the creatine phos- 
phoric acid to the above extent. The adenosin and the hexosephosphate 
molecules arc rejuvenated to about the same extent as those of the 
creatine phosphoric acid. Witli increasing temperature, as is to be 
expected, the rate of molecular rejuvenation increases, and in the course 
of less than a day practically all creatine phosphoric acid molecules are 
renewed. 

Phosphorus Isolated from Frog Killed 3 Hours after 
Subcutaneous In.tection of Labelled Sodium Phosphate 



Relative specific activity 
(activity per mgm V) 



Prog kept at 2° 



Inorganic P 

Creatine P 

Adenosin P (7 min hydrolysed at 100°) 
"Hexose" P (30 min hydroly.sed at 100°) 
Product of 100 min hydrolj'sis at 100" 
Non-acid soluble residual fraction . . 



Frog kept at 21° 




100 

78 

78 
38 



The new formation of some of the 'acid soluble' phosphorus compounds 
present in the blood also takes place to a very appreciable extent. In 
human blood 2 hours after intravenous injection of labelled sodium 
phosphate, the specific activity of the total acid soluble organic phos- 
phorus, kindly extracted by Mr. A. H. W. Aten from the blood cor- 
puscles, amounted to 20 per cent of that of the plasma inorganic phos- 
phorus. 

In experiments in vitro^'^Hn which dog's blood was shaken for 2.5 
hours with labelled sodium phosphate, 1/25 of the total acid soluble 
molecules was found to be labelled and thus split and resynthesized 
under the action of enzymes. In the same in vitro experiments the for- 
mation of only very minute amounts of labelled phosphatides (less 
than 0.1 per cent) could be ascertained. Also in experiments in vivo 
labelled phosphatides were found to be present to an appreciable extent 
in the blood only after much longer time. The specific activity of phospha- 
tide P extracted from human blood corpuscles 24 hours after administra- 
tion of labelled sodium phosphate was found to be 40 times less than 
that of plasma inorganic P, showing the very low rate of rejuvenation 
of the phosphatide molecules present in the blood. 

There is thus a conspicuous difference in the rate of rejuvenation of 
some low molecular water soluble compounds, as for example, creatine 
phosphoric acid, adenosin phosphoric acid, hexosephosphate, and 
non-w^ater soluble products like phosphatides, nucleoproteins and similar 



368 ADVENTURES IN RADIOISOTOPE RESEARCH 

compounds, present in the blood. This difference is closely connected 
with the fact that the first-mentioned compounds are at least partly 
rejuvenated through enzymatic action in the blood itself, while the latter 
are principally rejuvenated in the organs and carried from these in the 
blood stream. The measurement of the rate at which, for example, 
adenosintriphosphoric acid molecules are renewed can be conveniently 
used to determine the amount of enzymes the presence of which enables 
the exchange reaction to take place. 



References 

(DK.LoHMANN, Biochem. Z. 194, 306 (1928). 

<2) L. Hahn and G. Hevesy, Bull. Lab. Carlsherg 22, 188 (1938). 



Oi'ginally communicated in Kgl. Dannkc Videnskaberne-s Selskah. Biologiske 

Meddelelser, 15, 7 (1940) 

37. RATE OF RENEWAL OF THE ACID SOLI BLE 

ORGANIC PHOSPHORUS COMPOUNDS IN THE ORGANS 

AND THE BLOOD OF THE RABBIT 

G. Hevesv and L. Hahn 

From the Institute of Theoretical Physics, University of Copenhagen 

In a paper published recently in these Pioceeclings^i\ the rate of renewal 
of the phosphatide molecules present in various organs of the rabbit 
and other animals was discussed. In the present publication, data on the 
rate of new formation of acid soluble phosphorus compounds are commu- 
nicated. The acid soluble organic P compounds represent a great variety 
of chemically very different bodies: esters as, for example, hexosephos- 
phate, nucleotide compounds as adenosintriphosphate, phosphagen, and 
other compounds. These compounds^^) ^j-g renewed at a comparatively 
fast rate in the organs in contradistinction to the phosphatides and 
desoxyribo nudeoproteins^^) Furthermore, while the rate of new forma- 
tion of the phosphatides in the circulation is almost negligible, the 
acid soluble phosphorus compounds are renewed at a remarkable rate in 
the corpuscles. These facts justify the consideration of the acid soluble 
phosphorus compounds from our view-point as a definite group of the 
phosphorus compounds present in the body, 

EXPERIMENTAL METHOD 

Labelled P as sodium phosphate was administered by intravenous 
or subcutaneous injection to rabbits all through the experiment in order 
to keep the activity of the plasma inorganic P at a constant level. After 
the lapse of some hours or days, the animal was killed by bleeding. 
The fresh organs were placed in liquid air and w-ere extracted immedia- 
tely with cold 10 per cent trichloroacetic acid. The inorganic phosphate 

^1^ G. Hevesy and L. Hahn, Kgl. Danske Vidensk. Sehkah. Biol. Medd. 15' 
5 (1940). 

^-^ With the exception of adenylic acid [T. Korzybski and J. K. Pakxas, 
Z. physiol. Chem. 255, 195 (1938)] and, possibly, of other not yd known n>inor 
components of the acid soluble P mixture. 

^■''^ L. Hahn and G. Hevesy, Nature, April (>, 1940. 

"24 Heresy 



370 ADVENTURES IN RADIOISOTOPE RESEARCH 

present in the extract was precipitated as ammonium magnesium phos- 
phate. The filtrate obtained was then hydrolysed with 1 n HgSO^ for 7 
min at 100° to split off the labile P which was then precipitated as ammo- 
nium magnesium salt. The filtrate obtained after the last mentioned 
operation was hydrolysed 100 min to split off the phosphate radical 
of the hexosephosphate present. In order to avoid several consecutive 
precipitations of ammonium magnesium phosphate which lead to an 
accumulation of very appreciable amounts of ammonium salt in the 
soluble fraction, we usually divided the filtrate obtained after precipi- 
tation of the inorganic phosphate present as such in the tissue into 
aliquot parts. One aliquot part was hydrolysed for 7 min, the phosphate 
split off was precipitated, and the filtrate obtained was hydrolysed 
for 100 min. Another aliquot part was hydrolysed for 7 min, the filtrate 
obtained was hydrolysed for 12 hours, and so on. The phosphate of the 
creatinephosphoric acid was split off by heating the solution for 30 min 
to 40°. In some cases, the total acid soluble organic P w^as converted 
into phosphate and Mas investigated in toto. The ammonium magnesium 
phosphate precipitates obtained were dissolved in diluted hydrochloric 
acid and an aliquot part was used for a colorimetric P determination. 
To another aliquot part about 80 mgm non-active sodium phosphate 
was added ; the total P present in the solution was then precipitated 
as ammonium magnesium salt. The radioactivity of these precipitates 
was determined by the aid of a Geiger counter. 

Though the separation of the different acid soluble P compounds 
described above is far from being quantitative, it sufficed in most cases 
for our purpose. 

In the experiments with blood, as anti-coagulent, ammonium oxalate 
was used. The corpuscles w^ere centrifuged off and washed twice with 
a physiological sodium chloride solution. In experiments in vitro, the 
blood was kept in a COg— O2 atmosphere and was shaken, after addition 
of labelled sodium phosphate of negligible weight, for 30 — 190 min 
in a thermostat at 37°. 



RATE OF NEW- FORMATION 

As labelled phosphorus atoms can only be incorporated into organic 
molecules in the course of a synthetic process, the radioactivity of the 
organic phosphorus compounds isolated from an organ is a measure 
of the rate of its total or partial resynthesis. It is, however, not permitted 
to compare the specific activity (activity per mgm P) of the hexose- 
monophosphate extracted from the kidney and the muscle, for example, 
and to conclude from the fact that the hexosemonophosphate extracted 
from the kidney is much more active than that secured from the muscle, 



RENEWAL OF ACID SOLUBLE I'HOSPHORUS COMPOUNDS IX ORGANS OF RABBIT 371 

that the rate of new formation^i^ of hexosemonopliosphate is correspond- 
ingly larger in the kidney. The incorporation of labelled P atoms into 
the hexosemonophosphate molecules must be preceded by a penetra- 
tion of the labelled inorganics P into the cells of the organ. ]f this 
process is slow, the rate of formation of labelled hexosemonophosphate 
molecules is bound to be comparatively slow, in spite of a possibly 
very fast rate of new formation of the hexosemonophosphate molecules 
inside the cells of the organ in question. In fact, the labelled inorganic 
phosphate molecules penetrate very much faster into the kidney cells 
than into the muscle cells. To get proper information on the rate of 
renewal of an organic compound in an organ, we have to compare the 
specific activity of the P isolated from the organic compound in question 
at the end of the experiment with the average value of the specific activ- 
ity of the cellular inorganic P prevailing during the experiment. The 

Table 1. — Extent of Rexewal of the Total Organic Aciu .Soluble P in the 

Organs of the Rabbit 

Rabbit II. Weight 2.6 kgm. 
Inti'avenous injection during 215 min 





A 


B 


c 


D 








Specific 


Specific 


Average 


Specific 


~ X 100 


— X 100 




activity of 


activity of 


specific ac- 


activity of 





B 




the tissue 


the cellular 


tivity of the 


the organic 






1- g a u 


inorganic P 


inorganic P 


cellular P 


P 


Upper limit 
of the per- 


Lower limif. 
of the per- 




at the end 


at the end 


during the 


at the end 


centage 


centage 




of the ex- 
periment 


of the ex- 
periment 


experiment 


of the ex- 
periment 


renewed 


renewed 


Plasma 


100 
12.7 


12.7 


6.4 


12.7 


199 




Corpuscles 


100 


Kidney 


87.4 


87.1 


77.8 


33.6 


43.2 


38.6 


Small intestine, 














mucosa 


47.4 
44.0 


45.2 
40.6 


22.6 
20.4 


24.0 
14.3 


106 

70.2 


53.1 


Liver 


35.2 


Lungs 


36.5 


26.9 


13.4 


9.5 


71.0 


35.3 


Spleen 


30.8 
25.9 


28.5 
23.6 


14.3 

11.8 


6.9 


58.5 





Stomach 


29.2 


Heart 


25.5(1) 


21.4 


10.8 


8.6 


79.6 


40.2 


Brain 


1.32 


— 


— 


0.56 


— 










(') The inorganic P extracted from the heart contains partly such inorganic P atoms which were formed 
through decomposition of creatinephosphoric acid prior to the extraction. As the specific activity of the 
creatine P is, after the lapse of i hours, lower than that of the inorganic P (comp. the muscle values in Table 
3!), the specific activity of the cellular inorganic P of the heart is in fact higher than that stated above 
and, correspondingly, the values of the rate of renewal of the organic acid soluble P compounds in the lieart 
are smaller than those stated in the last and the last but one column of Table 1. 

^1^ The significance of the notion of the rate of now formation is discussed in 
the paper by G. Hevesy and L. Hahn, Kgl. Danske Vidensk. Selskab, Biol. Medd. 
15,5(1940). 



24" 



372 



ADVENTURES IN RADIOISOTOPE RESEARCH 



considerations mentioned above are discussed in detail in the publication 
cited above on the turnover rate of phosphatides. In this paper is de- 
scribed the method which permits us to calculate from the specific activity 
of the tissue inorganic P. the specific activity of the plasma inorganic P, 
the extracellular space of the organ, and the inorganic P content of the 



Tablio 2. — Extent of Renkwal of Diffeeent Fkactions of the Organic Acid 

Soluble P 

Rabbits II, III, and IV (average) 
Intravenous injection during 4 houre 









Spec, activity of the or- 


Spec, activity of the or- 








ganic P at the end of the 


ganic P at the end of the 








experiment : Average 


experiment : Specific 


r g ;» n 




Time of hydrolysis in 1 N 
HjSOj at 100° 


specific activity of the 

oelUilar inorganic P during 

the experiment. 

(Upper limit of the per- 
centage renewed) 


activity of the cellular 

inorganic P at the end 

of the experiment. 

(Lower limit of the per- 
centage renewed) 


Liver 


— 7 min 
non-hydrolysed 


152 

66 


76 


Liver 


> < . 


33 


Kidney, cortex .... 




0—100 min 


64 


57 


Kidney, cortex .... 


> • • 


100 min— 12 hr. 


47 


42 


Kidney, cortex .... 




non-hydrolysed 


29 


26 


Kidney, cortex .... 




hydrolysed in 1 N 
NaOH at 80° 


\ - 


43 



Table 3. — Specific Activity of Acid Soluble P Fractions 
Extracted from the Organs of the Rabbit 

Rabbit VII. Weight 2.4 kgm 
Subcutaneous injection during 11.5 hours 



Fraction 



Plasma inorganic P 

Corpuscle inorganic P 

Corpuscle P hydrolysed 15 hours in 1 N 112804 at 100' 
Corpuscle P hydrolysed 15—120 houi-s in 1 N HgSO^ at 10° 

Corpuscle non-hydrolysed residue 

Muscle inorganic P 

Muscle creatine P 

Marrow inorganic P(0 

Marrow organic P 

Brain inorganic P 

Brain organic P 



Specific activity 

at the end of the 

experiment 



100 
25 

25 

2.5 

13.0 

15.5 

8.5 

13.1 

36.8 

3.0 

2.3 



<') The low value is presumably due to the presence of traces of slightly active bone P in the marrow sample- 



RENEWAL OFACIDSOLIBLK PlIOSl'lIOIUS COMPOUNDS I.\ OIUJAXSOK RABBIT 378 

organ and plasma the average specific activity of the cellular inorganic 
P during the experiment. From the latter magnitude and the specific 
activity of the P of the organic phosphorus fraction at the end of the 
experiment, we can calculate what percentage of the organic compound 
in question is newly formed during the experiment, if only the extent 
of new formation is restricted. 

If a large fraction of the hexosemonophosphate molecules, for example, 
is newly formed during the experiment, we can no longer disregard the 
number of hexosemonophosphate molecules which were decomposed 
and resynthesized more than once during the experiment. If such a repeat- 
ed new-formation takes place, it will have the effect that the active 
hexosemonophosphate molecules present at the end of the experiment 
cannot be longer considered as having been formed with participation 
of inorganic P which had an activity corresponding to the average acti- 
vity prevailing during the experiment. The inorganic P atoms, which 
had an activity corresponding to a late stage of the experiment, will 

Table 4. — Specific Activity of Acid Soluble P Fractions 
Extracted from the Organs of the Rabbit 

Rabbit VIII. Weight 2.0 kgm 
Subcutaneous injection during 9 days 



Fraction 



Specific activity 

at the end of the 

experiment 



Plasma inorganic P 

Corpuscle total acid soluble P 
Muscle inorganic -j- creatine P 

Muscle ester P 

Brain inorganic -j- creatine P . 
Brain ester P 



100 
94 
40 
18.7 
18.8 
17.3 



Table 5. — Specific Activity of Acid Soluble P Fractions 
Extracted from the Organs of the Rabbit 

Rabbit IX. Weight 2.5 kgm 
Subcutaneous injection during 50 days 



Fraction 



Specific activity 

at the end of the 

experiment 



Plasma inorganic P 

Corpuscle total acid soluble P 
Muscle inorganic -(- creatine P 

Muscle ester P 

Brain inorganic -|- creatine P . 
Brain ester P 



100 

100 
88 
77 
56 
68 



374 ADVENTURES IN RADIOISOTOPE RESEARCH 

clearly be found to a larger extent incorporated in hexosemonophos- 
phate molecules than those P atoms the activity of which corresponds 
to an early stage of the experiment. 

Let us consider active hexosemonophosphate molecules which were 
formed during the first stage of the experiment and which were again 
newly formed during the last minute of the experiment. If the second 

Table 6. — Specific Activity of Acid Soluble P Fkactions 
Extracted from the Corpuscles of the Rabbit 

Rabbits II, III, and IV (average) 
Intravenous injection during 4 hours 



Time ot hydrolysis in 1 n H„S04 at 100" 



Specific activity 

at the end of the 

experiment 



Inorganic P (present as such in the corpxiscles) 

— 7 min 

7—100 min 

7 min — 12 hours 

Non-hydrolysed in the course of 12 hours (residue) 
Non-hydrolysed in the course of 24 hours (residue) 



100 
100 
100 
100 

87 
77 



process were not forthcoming, we should find molecules of small activity; 
if the opposite were the case, we should find the molecules to be strongly 
active. When calculating the fraction of the hexosemonophosphate 
molecules which were newly formed (once or several times) during the 
experiment from the ratio 

specific activity of h exosemonophosphate P a t the end of the experiment 
~ average specific activity of inorganic P during the experiment 

we overestimate the percentage of hexosemonophosphate which was 
renewed during the experiment. This will be especially the case if the 

ratio 

rate of renewal 

rate of intrusion 

is large, as, for example, in the case of the corpuscles. 
When calculating from the ratio 

specific activity of organic P at the end of the experi ment 
average specific activity of inorganic P during the experiment 

the extent of renewal of the acid soluble P mixture in the corpuscles, 
we arrive at a value of 199 per cent (see Table I). Such a calculation, 
for reasons stated above, supplies the upper hmit of the extent of renewal. 
The lower limit is given by the ratio: 



RENEWAL OF ACID SOLUBLE PHOSPHORUS COMPOUNDS IX ORGANS OF RABBIT 375 



specific activity of organic P at the end of the experiment 



specific activity of inorganic P at the end of the experiment. 

The actual value clearly lies very mucli nearer 1o the lower than to the 
upper limit. 

Tahlk 7. — ExPERiMKXTs in vitro With Hauhit JSi.ooj) 



Corpuscle fraction 



Duration of the 
experiment 



Specific activity 

at the end of tlie 

experiment 



Hydrolysed 7 min 

Hydrolysed 7 min — 12 hours 

Non-hydrolysed 

Inorganic P 

Hydrolysed 7 min 

Hydrolysed 7 min — 12 hours 

Non-hydrolysed 

Inorganic P 

Hydrolysed 7 min 

Hydrolysed 7 min — 12 hours 

Non-hydrolysed 

Inorganic P 

Organic acid soluble P 




16 
13 

100 
90 
41 
28 

100 
82 
57 
46 

100 



Table 8a. — Effect of Temperature on the Distribution of ^^P 
BETWEEN Plasma and Corpuscles 

Rabbits blood after addition of labelled phosphate of negligible weight 

is shaken for 90 min 





Temperature 




37° 5° 


Plasma inorganic P 

Corpuscle inorganic P 

Corpuscle organic P 


78 
3.8 
18.2 


9.67 
0.62 
2.64 



Table 8b. — Effect of Temperatxtre on the Distribution of '^p 

BETWEEN Plasma and Corpuscles 

Relative specific activity of the P fractions of the blood 



Fraction 



Temperature 



7° 



Plasma inorganic P 

Corpuscle inorganic P 

Corpuscle pyrophosphate P . 
Corpuscle non-hydrolysed P 




100 
0.37 
0.36 
0.087 



376 ADVENTURES IX RADIOISOTOPE RESEARCH 

Table 8c. — Effect of Temperature on the ^^P Fractions 

OF the Corpuscles 

Relative specific activity of the P fractions of the corpusclas 



Fraction 


Temperature 


37" 


5» 


Corpuscle inorganic P 

Corpuscle pyrophosphate P 

Corpuscle non-hydrolysed P 


100 

82 
50 


100 
84 
23.5 



DISCUSSION 

A. Renewal of the acid soluble P compounds in the organs. 

B. Renewal of the acid soluble P compounds in the corpuscles. 

A) Renewal of the acid soluble P compounds present in the organs 

As seen in Table 1, in the course of 4 hours a very appreciable part 
of the average acid soluble P compounds present ^n many of the organs 
was renewed. A very active turnover takes place in the mucosa of the 
small intestine. One half or more of the molecules of the organic acid 
soluble P compounds present in this organ became renewed in the course 
of 215 min. This very marked rate of new formation of the organic acid 
soluble P compounds is of interest in connection with the view put 
forward by Veezar and others on the role of intermediary phosphoryl- 
ation processes in the resorption of sugar from the intestine^^^.The highest 
value for the specific activity of the acid soluble oiganic P was found 
in the kidneys. The labelled inorganic P diffuses faster into the cells 
of the kidneys than into those of any other organ. The high value of the 
specific activity of the acid soluble kidney P is, to some extent, due to 
the fact that the cellular inorganic P Mithin 215 min acquires a higher 
value in the kidneys than in other organs. If due regard is taken to this 
phenomenon we find that, in spite of the fact that the specific activity 
of the intestinal acid soluble P is lower than that of the corresponding 
fraction extracted from the kidneys, the rate of renewal in the intestinal 
mucosa is greater than in the kidneys. 

The rate of renewal of the organic acid soluble P molecules in t he 
liver and in the lungs (see Table 1) is also quite appreciable. The compa- 
ratively high value found for the ratio of the specific activities of the 
organic P and inorganic P in the case of the brain tissue is, at least to 

(i) F. Verzar and E. J. McDougall, Absorption from the Intestine. London 
(1936). Comp. also E. Lundsgaaud, Z. physiol. Chem. 261, 19 (1939). 



RENEWAL OF ACID SOLUBLE PHOSPHORUS COMPOUNDS IN ORGANS OF RABBIT 377 

some extent, due to an extremely low activity of the average inorganic- 
P of the brain. It is a puzzling result that the total activity found in 
the brain tissue, due to the presence of active inorganic and organic P, 
is smaller than that we should expect to find in the interspaces of the 
brain alone when assuming a proportional distribution of the active 
inorganic P between the plasma and the extracellular space of the brain 
tissue. In this calculation, the extracellular space is taken to be 30 per 
cent of the weight of the brain, as found from the distribution figures, 
of chlorine and sodium^^) between the plasma and the brain tissue. Our 
results suggest the assumption that the labelled phosphate ions penetrate 
at a very slow rate through the capillaries of the brain or, alternatively,, 
that the figures obtained by determining the distribution of chlorine 
of sodium between the plasma and the brain do not represent the proper 
extracellular space of the brain. It is for these reasons that we did not 
state in Table 1 any figures for the rate of renewal of the acid soluble 
P compounds present in the brain. 

Table 2 contains data on the activity of different organic P fractions 
extracted from the kidneys and the liver. The phosphate obtained after 
7 min hydrolysis contains, as well-known, besides P split off from 
ereatinephosphoric acid, the labile P of the adenosintriphosphate mole- 
cules. That the adenosintriphosphate molecules present in the muscle 
are reorganised at a fast rate was found in our previous experiments^^) 
Meyerhof and his collaborators^^^ studied the rate of reorganistion of 
the adenosintriphosphate molecule with incorporation of active inorganic 
P in experiments in vitro and found this process to take place at a very 
fast rate. Data on the activity of the phosphorus obtained by hydro- 
lysing the organic acid soluble phosphorus extracted from perfused 
cat liver for 7 min are given by Lundsgaard^*\ 

Our experiments lead to the result that at least 76 per cent of the 
7 min product extracted from the liver of the rabbit became renewed 
in the course of 215 min. In Lundsgaard's perfusion experiment, the 
specific activity of the 7 min fraction was found, after 90 min, to amount 
to 60 per cent of that of the inorganic P extracted from the plasma at 
the end of the experiment. 

As seen in Tables 2—7 the more readily hydrolysable compound is 
renewed at a faster rate than the less readily hydrolysable one. That 
even those compounds which resist treatment with 1 N HgSO^ at 100° 
for 12 hours or more are renewed, however, at a very appreciable rate 

<i>J. F. Manery and B. Hastings, J. Biol. Chem. 127, 657 (1939). 
(2) G. Hevesy and O. Rebbe, Nature 141, 1097 (1938); G. B-EVESY, Enzymologia 
5,138(1938). 

<') O. Meyerhof, P. Ohlmeyer, W. Gentner and H. Maieb-Leibnitz, Biochem. 
Z. 298, 398 (1938). 

We. Lundsgaabd, Skand. Arch. /. Physiol. 80, 291 (1938). 



'MH ADVENTURES IN RADIOISOTOPE RESEARCH 

is seen in Tables 2 and 3. More than 1/4 of the non-hydrolysable residue 
of the organic acid soluble P fraction secured from the kidneys was, 
for example, found to be renewed in the course of 215 min (see Table 2). 
After the lapse of so long a time as 9 and 50 days (see Tables 4 and 5), 
the muscle inorganic -f creatine P has only reached 40 and 88 per cent, 
respectively, of the specific activity of the plasma inorganic P. After 
the lapse of 50 days, the specific activity of the ester P of the muscles 
was found to be 77 per cent of that of the plasma inorganic P. A detailed 
investigation of the rate of renewal of the acid soluble P compounds 
present in the muscles of the frog will be published shortly. 

B) Renewal of the acid soluble P compounds present in the corpuscles 
1. Phosphorylation processes going on inside the corpuscles 

In our early investigations^^^ on the circulation of phosphorus, using 
radioactive P as an indicator, we found that the organic acid soluble 
P compounds of the red blood corpuscles are normally in a state of flux, 
being continuously decomposed and resynthesized. Labelled phosphate 
ions were found to penetrate into the corpuscles at a fairly slow rate 
and to take part in very rapid phosphorylation processes inside the 
corpuscles. Labelled hexosemonophosphate introduced into the plasma 
was found not to penetrate at any significant rate into the corpuscles. 
However, the labelled phosphate present in such hexosemonophosphate 
molecules after being split off diffuses as inorganic phosphate into the 
corpuscles and is incorporated inside the erythrocytes partly into hexose- 
monophosphate molecules. Presumably, the P atoms of the plasma 
diffuse exclusively or almost exclusively as phosphate ions into the 
corpuscles. 

That phosphorus compounds, as hexosephosphoric acid, triosephos- 
phoric acid, phosphopyruvic acid, phosphoglyceric acid, and so on, 
take an important part in glycolytic processes going on in the corpuscles 
was emphasised by v. Euler and Brandt^-^ and others. According to 
the views of Meyerhof, Parnas, and others, in the course of the gly- 
colytic cycle, hexosediphosphate, for example, is found to be formed 
through the interaction of dextrose with adenosintriphosphate. Hexose- 
diphosphate is maintained in enzymatic equilibrium with two molecules 

(^)L. Hahn and G. Hevesy, C. R. Lab. Carlsherg 22, 188 (1938). G. Hevesy 
and A. H. W. Aten, Kgl. Danske Vidensk. Selskab, Biol. Medd. 14, 5 (1939). 

(2> H.V.EuLEB and K. M. Brandt, T. physiol. Chem. 240, 215 (1936). Comp. 
also H. Lawaczeck, Biochem. Z. 145, 351 (1924); Negelein, Biochem. Z. 158, 
121 (1925); M. Martland, Biochem. J. 19, 117 (1925); P. Rona and K. Iwasaki, 
Biochem. Z. 184, 318 (1917); H. K. Barrenscheen and B. VAsArhelyi, Biochem. 
Z. 230, 330 (1931); H. K. Barrenscheen and K. Braun, Biochem. Z. 231, 144 

r(1931). 



RENEWAL OF ACID SOLUBLE PHOSPHORUS COMPOUNDS IN OKGANS OF RABBIT 379 

of triosephophate. The last mentioned compound reading with pyruvic 
acid forms phosphoglyceric acid which is converted into phosphopyruvic 
acid and this, in turn, reacts with adenylic acid in tlie resynthesis of 
adenosintriphosphate. The last mentioned compound is also formed by 
direct phosphorylation of adenylic acid from inorganic phosphate or by 
transfer of the phosphate radical of glycerophosphate to adenylic acid. 
The synthesis of adenosintriphosphate is a very rapid process and the 
active inorganic phosphate ions which penetrate into the corpuscles 
will soon be found to be incorporated in adenosintriphosphate molecules. 
The participation of the active adenosintriphosphate molecules in the 
synthesis of various organic P compounds will lead to the formation 
of active hexosephosphate, active phosphoglyceric acid, and so on, 
in the corpuscles. In this connection, the result obtained by Dische^^^ 
is of interest : he found that the total phosphate transferred to glucose 
added to human erythrocytes originates from adenosintriphos- 
phate. 

Important evidence that the organic acid soluble phosphorus com- 
pounds and, primarily, diphosphoglycerate of the red blood corpuscles 
constitute a labile phosphorus reserve of considerable consequence, 
serving various functions was presented in recent years by Guest and 
his colleagues®. Some of their findings are described in what follows. 

The development of rickets in rats is associated with decreases in 
all fractions of the acid soluble phosphorus. During the first five days, 
the concentration of inorganic phosphorus and adenosintriphosphate 
phosphorus drops abruptly to a low level and then remains constant 
for 25 days and longer. The decrease in the organic acid soluble phos- 
phorus is accounted for almost entirely, after the first few days, in the 
diphosphoglycerate fraction. Guest and Rappaport state that diphos- 
phoglycerate makes out about half of the acid soluble phosphorus pre- 
sent in the corpuscles. 

In experiments carried out on dogs after nephrectomy, it was found 
that, due to the failure of excretion of the vaste endogenous P, a large 
increase in the inorganic P content of the blood takes place, which is 
followed by a corresponding increase in the acid soluble organic P content 
of the corpuscles. The increase is mainly due to the rise of the diphospho- 
glycerate content of the corpuscles, the increase in organic acid soluble 
P and in diphosphoglycerate P being 47 and 43 mgm, respectively, 
per hundred cc. 

They found, furthermore, that the increase of phosphorus excretion 
in the urine during acidosis comes partly from mobilised diphospho- 



iZ. DiscHE, Naturwiss. 24, 462 (193G). 

(2) A summary of many of their results is to be found in tlic^ paper by G. M. 
Guest and S. Rappapokt, Amer. J. Dis. Children 58, 1072 (1939). 



380 ADVENTURES IN RADIOISOTOPE RESEARCH 

gly cerate of the corpuscles. As an effect of pyloric obstruction, an in- 
crease of the acid soluble P content amounting to 37 mgm. equiv. per kgm 
corpuscle water of the clog was found to take place. From this increase, 
32 mgm equiv. were due to the rise in the glycerophosphate content. 

These and numerous other findings clearly show that the acid soluble 
phosphorus compounds of the red corpuscles are readily synthesised 
and decomposed in the blood through reactions of the glycolytic cycle. 
That these processes take place in the corpuscles at a remarkable speed 
was shown by us when making use of radioactive phosphorus as an 
indicator. We have, thus, two independent lines of evidence as to the 
remarkably high rate of turnover of phosphoglycerate and some other 
phosphorus compounds present in the corpuscles. 

By comparing the specific activity of the inorganic P of the corpuscles 
with that of the P extracted from various organic compounds present 
in the corpuscles we get information on the rate of resynthesis of these 
compounds. The comparison of the specific activity of the inorganic P 
present in the corpuscles with that of the inorganic P present in the 
plasma informs us, on the other hand, on the rate of penetration of 
phosphate ions from the plasma into the corpuscles. 

2. Rate of new formation of the acid soluble P compounds present 
in the corpuscles 

As seen in Table 7, which gives the result of experiments in vitro, 
the product of 7 min hydrolysis has, after the lapse of 30 min, a specific 
activity amounting to 77 per cent of that of the corpuscle, inorganic P. 
The product hydrolysed between 7 min and 12 hours, which contains 
besides hexosephosphate P and other fractions appreciable amounts 
of diphosphoglycerate P as well, is markedly less active than the readily 
hydrolysed fraction, while the specific activity of the P of the non- 
hydrolysed residue is only 1/8 of that of the corpuscle inorganic P. 
This fraction^^^ consists mainly of 2, 3-diphosphoglyceric acid P though 
it contains also P of the adenylic acid which amounts, in the corpuscles 
of the rabbit, to about 5 — 10 mgm per cent, thus to about 1/10—1/20 
of the total acid soluble P of the corpuscles. 

In experiments in vivo taking about four hours, all but the non- 
hydrolysed fraction were found to be entirely renewed; only about 
1/5 of the last mentioned fraction, presumably mainly its adenylic acid 



(i) E. Greenwald, J. Biol. Chem. 63, 339 (1925); H. Jost, Z. phi/siol. Chem. 
116, 171 (1927); S. E. Kerr and A. Autaki, J. Biol. Chem. 121, 531 (1927); E. 
Wabweg and G. Stearns, J. Biol. Chem. 115, 567 (1936); S. Rappaport and 
G. M. Guest, J. Biol. Chem. 129, 781 (1939); A. Lennerstrand and M. Lenner- 
STRAND, Ark. Kemi, 13, B, No. 15 (1939). 



RENEWAL OF ACID SOLUBLE PHOSPHORUS COMPOUNDS IN OKiiAXS OF RABBIT liH 1 

content^i-, was found to be unchanged. Diphosphoglycoric acid is, llius, 
renewed at a high rate as well. 

Rate of penetration of plasma inorganic P into the corpuscles 

To obtain information on the rate of penetration of the inorganic 
phosphate of the plasma into the corpuscles, w-e have to compare the 
specific activity of the plasma inorganic P with that of the corpuscle 
inorganic P. After the lapse of 11.5 hours (see Table 3), this ratio is 
found to be 4, showing that the rate of penetration of the phosphate 
ions from the plasma into the corpuscles and vice versa is slows a much 
slower process than the reorganisation of most of the acid soluble organic 
P compounds present in the corpuscles. After the lapse of nine days, 
the ratio of the specific activity of the plasma inorganic P and the cor- 
puscle average acid soluble P is only slightly larger than 1 (1.06) (after 
so long a time, the activity of the average corpuscle acid soluble P acqui- 
red almost the same value as shown by the inorganic P of the corpuscles); 
and after the lapse of fifty days, a completely proportional distribution 
of the labelled P atoms between the plasma P and the P of the acid 
soluble P compounds present in the corpuscles is attained. While, after 
the lapse of 11.5 hours, the chance of a normal distribution of a P atom 
which diffused into the corpuscles between organic and inorganic P is 
almost 1, the corresponding figure for the distribution of an inorganic 
P atom between plasma and corpuscles is only of the order of magnitude 
of 1/4. 

The interesting phenomenon that an individual phosphate ion, while 
penetrating fairly slowly into the corpuscles, is incorporated at a remark- 
ably fast rate into organic molecules present in the corpuscles, finds 
many analoga in the processes going on in various organs. It is especially 
conspicuously shown in the study of the penetration of labelled phosphate 
into the muscle cells and of that of the rate of renew-al of the acid soluble 
P compounds present in these cells; the former process being slow, the 
latter process being, in the case of some of the compounds, very fast. 

It is very probable that a large part of the P atoms present 
in the molecules, of most of the acid soluble organic P compounds 
of the corpuscles, were incorporated into these molecules inside the 
corpuscles and reached the erythrocytes as inorganic phosphate ions 
which passed from the plasma into the corpuscles. The possibility that 
hand in hand with the process mentioned above a slow exchange of, 
for example, organic phosphoglycerate between plasma and corpuscles 

^i) S. E. KERBandL. Daoud [J. Biol. Chem. 109, 304 (1937)] state that, out 
of 88 mgm por oont organic acid soIuVjIo P found in tho coipusolos of tho labbit, 
IG mgm per cent arc pyropliospliatc P and 8 mgni per cent adenylic acid P. 



382 ADVENTURES IN RADIOISOTOPE RESEARCH 

takes place cannot be disregarded. In view of the very low content of 
organic acid soluble P compounds of the plasma, if a migration of such 
compounds between corpuscles and plasma would take place, it should 
be mainly directed from the corpuscles into the plasma. In view of 
the fast rate of renewal going on in the corpuscles and the fast turnover 
of the acid soluble P compounds in the plasma, the investigation of a 
migration of organic acid soluble P molecules from the corpuscles into 
the plasma or vice versa encounters difficulties. 

In the above connection it is of interest to remark that Solomon, 
Hald and Peters^'^ found, in a recent investigation, that phosphate 
esters present in the corpuscles are restrained from escaping by some 
force in addition to the membrane of the corpuscles. The restraining 
force is presumably a chemical aggregation or combination with sub- 
stances of large molecular size. They arrived at the result mentioned 
above by the following observation. When filtering blood which was 
hemolysed by freezing, the ultrafiltrate obtained at 7° did not contain 
any appreciable amount of organic P, while the opposite was the case 
when saponin was used to obtain hemolysis. Frozen blood acts as much 
as does intact blood so far as phosphates are concerned. The organic 
esters remain intact as long as the blood is kept cold and their combi- 
nation with substances of high molecular size remains unpaired. This is 
not the case when saponin is added. Under the action of this agency the 
binding forces break down, and the organic phosphate esters can enter 
the ultrafiltrate. At 37° the phosphate esters can be ultrafiltrate d even 
if the blood was hemolysed by freezing. In experiments in vitro with 
intact blood at 37°, during 18 hours no appreciable amount of organic 
phosphate ester was found to escape from the corpuscles into the plasma. 
These results support the view that the P atoms present in the phosphate 
ester molecules of the corpuscles reach the plasma, and vice versa, 
after being converted into constituents of organic phosphate ions. 

As seen in Tables 8 a, b and c, with decreasing temperature the rate 
of penetration of ^^P from the plasma into the corpuscles and also the 
rate of its incorporation into organic P compounds strongly decreases. 
While at 37°, in the course of 90 min, 22 per cent of the ^^P originally 
present in the plasma diffused into the corpuscles, at 5° only 3.3 per 
cent of the ^^P originally present in the plasma found their way into 
the corpuscles. The comparison of the specific activity of the inorganic 
P present in the corpuscles at 37° and 5°, respectively, leads to the 
result that this activity is 37 times larger at 37° than at 5°. A similar 
comparison of the specific activity of the organic P of the corpuscles 
(exclusive the labile P of adenosintriphosphate) leads to a ratio of 80. 
It is of interest to note that a decrease of the temperature hardly affects 

'^^F. C. Solomon, P. M. Hald and J. P. Peters, J. Biol. Chem. 132, 721 (1940). 



KEXEWAL OF ACID SOLUBLE PHOSPIIOKIS COMPOUNDS I.\ OUGAXS OF RABBIT 383 

the very fast rate of new-formation of adenosintriphosphatc molecules^ 
since more than 80 per cent of the labile P of the adcnosintriphosphate 
present in the corpuscles became renewed during the experiments bolli 
at high and at low temperatures. 

Summary 

Labelled phosphate was administered to rabbits all through the experiments- 
in order to keep the activity of the inorganic phosphate of the plasma at a constant 
level. The experiments took 215 min to 50 days. The comparison of the specific 
activities of the organic P and the cellular inorganic P extracted from the organs 
leads to the result that in the course of 215 min more than one-half of the acid 
soluble P compounds present in the mucosa of the small intestine became renewed. 
Next the intestinal mucosa, the fastest rate of turnover was found to take place 
in the kidneys, liver and lungs. 

From the various organic acid soluble phosphorus compounds the most readily 
hydrolysable ones were found to be renewed at the fastest rate. Fractions contain- 
ing mainly phosphoglycerate were found to be renewed at an appreciable rate 
as Avell. 

While the rate of formation of labelled acid soluble organic P compounds 
inside the corpuscles is rapid, the diffusion of labelled phosphate ions from the 
plasma into the corpuscles is a slow process. In the course of 12 hours, only about 
1/4 of the P atoms of the acid soluble phosphorus compounds of the corpuscles 
entered into exchange equilibrium with the P atoms of the plasma phosphate, 
while most of the molecules of the compounds mentioned above were renewed 
during this time inside the erythrocytes. The contrast between the rate of inter- 
penetration of labelled phosphate and that of its incorporation into several of the 
acid soluble phosphorus compounds inside the cells is also fovmd in the case of 
the muscles. 

Labelled phosphate was found to penetrate into the brain tissue at an exceed- 
ngly low rate. 



Oiiginally communicated in Kgl. Danske VidensJcabernes Selskab. Biologiske 

MeddeleUer, 16, 8 (1941) 

38. CIRCULATION OF PHOSPHORUS IN THE FROG 

G. Hevesy, L. Hahn and 0. Rebbe 

From the Institute of Theoretical Physics and the Zoophysiological Laboratory, 

University of Copenhagen 

This paper contains the description of some experiments on the circu- 
lation of phosphorus in the frog using racliophosphorus as an indicator. 
The experiments described were carried out in the course of the last 
six years^^). 



EXPERIMENTAL PROCEDURE 

Most of the radioactive phosphorus (^^P) used in these experiments was pre- 
pared by irradiating carbon disulphide with neutrons emitted by a radium-beryl- 
Hum source. The ^^p produced was extracted with diluted nitric acid and turned 
into sodium phosphate. This sodium phosphate of negligible weight (10""io gm 
or less) was dissolved in 0.6 per cent sodium chloride solution. A few tenths of a 
cubic centimetre of the solution were injected into the lymph sack of Rana escu- 
lenta or Rana hungarica. In our earlier experiments, the injection took place 
at the start of the experiment; in our later work, however, we injected labelled 
phosphate several times during the experiment in order to keep the activity of 
the plasma at an approximately constant level. Experiments were carried out 
both at 0° and at about 20°; their duration varied between 5 minutes and 400 
hours. The muscle tissue was put into liquid air immediately after its removal 
and the acid soluble constituents were extracted with cold 10 per cent trichloro- 
acetic acid. The filtiate obtained was at once added to a solution contaraing ammo- 
nia and magnesium citrate. By this procedure, all inorganic P present in the tri- 
chloroacetic extract was precipitated. The next step was to hydrolyse the creatine- 
phosphoric acid present in the filtrate. In our early work, we decomposed the 
creatine phosphoric acid by adding anunonium molybdate and keeping the solution 
for 1 hour at 40° in 1 N. HgSO^. Later, we omitted the addition of ammonium 
molybdate and split off the phosphate by letting the acidified solution boil for 
a short time, as proposed by Meyerhof et a/ia.^2)xhe phosphate group, split off 
from the creatinephosphoric acid, was then precipitated as ammonium magnesium 



^1^ O. Rebhk, M. Sc, who has taken the deepest interest in the problems dis- 
cussed in this paper and worked for several years almost incessantly on their 
elucidation suffered an untimely death the 5th of December, 1940. 

^-^ O. Meyerhof, P. Ohlmeher, W. Gentnek and H. Maier -Leibnitz, Biochem. 
Z. 298, 400 (1938). 



CIRCULATIOX OF PHOSPHORUS IN THE I'llOG 385 

salt. The filtrate thus obtained was acidified and the 1 N. HgSO^ solution obtained 
was boiled for 7 minutes in order to split oil' the pja-ophosphate group of the 
adenosintriphosphate. In other experiments, the solution was kept at 100° for 
100 minutes. By this procedvire, the hoxosemonophosphate was liydrol>sed. 
Other fractions were secured by hydrolysing the filtrate for some houi-s. The 
non-hydrolysed phosphorus compounds remained in the filtrate together with 
large amounts of neutral salts. From this solution, after wet ashing of the organic 
compounds present, the phosphorus was precipitated as molybdenum compound. 
The ammoniuni phosphomolybdate was dissolved in ammonia, and phosphorus 
was precipitated as ammonium magnesium phosphate. 

The muscle fraction which does not dissolve in trichloroacetic acid contains 
the phosphatides and the residual P. To secure the phosphatides, Bloor's method 
was used. All fractions were ultimately obtained as ammonium magnesium phos- 
phate which was dissolv^ed in dilute hydrochloric acid. An aliquot part of the 
solution was used for the colorimetric determination of the phosphorus, accortling 
to FiSKE and Subbarow, another aliquot part was applied in the radioactive 
measurements. The comparison of the radioactivity of the P fractions is much 
facilitated if all samples have the same weight. In this case, no correction for the 
absorption of the ^-rays in the sample has to be made. Fractions of equal weight 
were obtained by adding to the solution of each fraction 80 mgm of secondary 
sodium phosphate and by precipitating all P present as ammonium magnesium 
phosphate. The precipitates obtained were dried at 106°. Corrections for the decay 
of the activity of the ^-P can also be avoided if all samples are measured relatively 
to the same active phosphorus preparation. As such preparation an aliquot of 
the solution administered was used, the P content of the preparation being con- 
verted into ammonium magnesium phosphate, as described above. 



DISTRIBUTION OF PHOSPHORUS IN THE FROG 

We determined the phosphorus content of the different parts of the 
frog by wet ashing of the organs followed by a phosphorus determination 
by the method of Fiske and Subbarow. The results of these determina- 
tions are seen in Tables 1 a and 1 b. 

ABSORPTION OF PHOSPHATE 

In order to get data on the rate of absorption of the lal)elled phosphate 
injected into the lymph sack, we determined the activity of plasma 
samples of known weight both of frogs kept at 0° and frogs kept at 
18° at different times after injecting the labelled phosphate. The figures 
obtained (see Figs. 1 and 2) are not a direct measure of the amount 
of 32p absorbed but indicate the difference between the amount absorbed 
into the circulation and the amount which left the circulation for the 
organs. (The amount taken up by the corpuscles in the course of a few 
hours is very restricted; see p. 400.) 

2.^ lievesy 



386 



ADVENTURES IN RADIOISOTOPE RESEARCH 



Table la. — Distribittion of Phosphorus 
IN Rana csculenta Weighing 58 gm 



Organ 


intrm 1' 


Per cent 
of total P 


BloocKi) 


0.83 

31.50 

59.32 

335.30 

4.28 
20.61 


0.18 


Skin 


6.96 


Muscles 

Bones 

Liver(2) 


13.20 

74.16 

0.94 


Remaining part 


4.56 


Total 


451.84 





'■> E.xtrapolated value, assuming the blood content to constitute 4 per cent of the weight of the frog. 
'"' The P content of the liver phosphatides was found to be 22 per cent of the total P content of the liver. 



Table 16. — Distribution of Phosphorus 
IN the Muscle of the Frog 



Fraction 


Per cent of total 
P content 


Acid soluble P 

Phosphatide P 

Residual P 


82.1 

10.8 

7.1 




3 4 

Time in hours 



Fig. 1. ^-P content of the frog's plasma after injecting ^-P into the 

lymph sack. T = 0°. 



CIRCl'LATION OF IMIOSI'HORUS IX THE FKOO 



387 



Temp -21 



^No 





2 5 

Time in hours 



Fio. 2. 3'-P content of the frog's plasma after injecting ^'P into the 

lymph sack. T = 21''. 



In another set of experiments, we injected large amounts of phosphate 
(corresponding to 6.1 mgm P) into the lymph sack of the frog. The 
inorganic P content of the plasma at different times is seen in Table 2. 



Table 2. — P Content of the Plasma after Injecting 6.1 mgm. 
P into the Lymph Sack of the Frog 



Time in hours 


Weight of the frog 
iu gm 


P content of the 
plasma mgm per cent 


Excess 1' present iu 

the pla.sma mgm 

per cent 


0.5 

1.5 

19.0 


76 

78 
50 


21.7 
23.9 
10.3 


18.1 

20.3 

6.7 



RELATIVE RATE OF UPTAKE OF LABELLED PHOSPHORUS BY THE 

BONE AND THE MUSCLE 

In our preliminary experiments, the results of which are reported 
in Tables 3 — 10, we compared the uptake of labelled phosphorus by 
fresh bone and muscle samples of equal weight at 2° and 22°, respect- 
ively. 1 gm muscle tissue was found to take up less ^^p than 1 gm bone 
tissue, though the difference is much less than would be expected if 



25* 



388 



ADVENTURES IX RADIOISOTOPE RESEARCH 



Table 3. — Frog I, Kept at 2°, Killed after 12 Days 
Ash weight: 3.235 gm 









Per L-ent liilielleil P 




Weight of fresh tissue 


Weight of ash 


aclmuiistered found 
in 1 gm fresh tissue 






Bones 






mgm 


mgm 




Right femur 


427.8 


126.3 


4.33 


Left femur 


442.8 


123.6 


4.74 


Right tibia 


469.5 


149.2 


4.76 


Left tiVjia 


432.6 

gm 


141.1 
Muscle s 


5.23 


Right Ihigh 


3.989 




0.284 


Left thigh 


3.467 


— 


0.262 


Right calf 


1.401 


— 


0.237 


Left calf 


1.336 


— 


0.199 



1 mgm bone P had the same chance to be replaced by labelled P as 
1 mgm muscle P. 1 gm bone tissue contains about 30 times as much 
P as 1 gm muscle tissue. The comparatively low activity of the ])one P 
is due to the fact that those phosphorus ions which are located on the 

Table 4. — Frog II, Kept at 2°, Killed after 12 Days 
Ash weight: 3.014 gm 



Weiglit of fash tissue 



Weight iif ash 



Per cent of labelled 
P administered found 
in 1 gm fresh tissue 



Right femur 
Left femur . 
Right tibia . 
Left tibia . . 

Right thigh . 
Left thigh . . 
Right calf . . 
Left calf . . . 



gm 

5.461 
4.635 
1.670 
1.679 





B 


ones 




mcrm 




mem 




417.0 




123.9 


4.74 


425.4 




116.1 


4.38 


450.6 




1.50.8 


5.01 


450.7 




1.J2.2 


4.74 




M 


u s c 1 e s 





0.24 
0.23 
0.28 
0.31 



surface of the apatite crystallites containing the mineral constituents 
of the bone can be replaced by a physical exchange process with the 
labelled phosphate in the plasma or the lymph, while the phosphate 
ions present inside these crystals cannot be replaced. Labelled phosphate 
ions can only be incorporalerl into the inside of the apatite crystalHtes 



CIRCULATIOX OF PHOSPHORUS IN THE FROCi 



389 



Tablk 5. — Froo TII, Kept at 2°, Killkd aktkh 21 Days 
Ash wciglit: 3.104 gm 



VVeiirlil of tresh tissue Weight oi asli 



Right tibia epiphysis 
Left tibia epiphysis . 
Right tibia diaphysis 
Left tibia tliaphysis . 

Right euir 

Left calf 



1.269 
1.252 



Per cent ut ImIjcHciI 

V ailministered found 

in 1 gm fresh tissue 





B ones 




mi<m. 


m^m 




155.1 


29.2 


6.03 


142.9 


29.1 


5.90 


205.7 


77.0 


6.34 


200.4 


7(5.1 
Muscles 


6.50 



0.42 



(luring the formation of such crystallites from a plasma containing 
labelled phosphate. The dissolution of "old" apatite crystals and the 
formation of "new" ones is, however, a comparatively slow process 
and is restricted to a fraction of the bone apatite. In contrast to the 



Table 6. — Frog IV, Kept at 2°, Killed after 22 D.ws 
.\sh \\'eight: 3.347 gm 



W eisjt of fresli tissue 



j Per cent o£ laholled P 
Weight of ash | administered found 
in 1 gm fresh tissue 



Right tibia epipliysis 
Left tibia epiphy.sis . 
Right tibia diaphysi.s 
Left tibia diaphysis . 

Right thigh 

Left thigh 

Right calf 

Left calf 



mgm 

144.2 

158.7 

205.8 

215.2 

4.116 
4.109 
1.477 
1.548 



Bones 


mgm 


33.1 


39.6 


86.6 


80.8 


M n s c 1 e s 



3.6S 
4.50 
5.07 
4.52 



(t.2() 
0.29 
0.34 
0.36 



bones, the greatest part of the phosphorus in the muscles is present as 
a constituent of organic compounds, as seen in Table 1 b. The bulk 
(about 80 per cent) of the organic P is prescnl in llie muscle of the 
frog in the form of acid soluble phosphorus compounds and, in a 
corresponding mann(>r, the ia1e of adivation of the muscle P depends 



390 



ADVENTURES IX RADIOISOTOPE RESEARCH 



mainly upon the rate of formation of active acid soluble P compounds. 
This process is much faster than the formation of the apatite crystals 
of the bone tissue and this fact explains why the replacement of 
phosphorus in the muscle tissue takes place at a much more rapid rate 



Table 7. — Frog V, Kept at 20 — 24°, Killed after 8 Days 

Ash weight: 3.65 gm 



Weight of fresh tissue Weight of ash 



Per cent of labelled P 
administered found 
in 1 Km fresli tissue 



Riglit femur epiphysi.s 
Left femur epiphysi.s 
Right femur diaphysis 
Left femur diaphysis 
Right tibia epiphysis 
Left tibia epiphysis . 
Right tibia diaphysis 
Left tibia diaph^'sis . 



Right thigh 
Left thigh . 
Right calf . 
Left calf . . 



mgm 

172.lt 

183.9 

412.7 

436.4 

265.9 

237.1 

374.4 

358.7 

gm 
5.372 
5.632 
L925 
1.980 



Bones 
mtrm 

33.5 

36.9 
123.5 
123.9 

59.6 

62.4 
141.9 
130.2 

Muscle s 



3.30 
3.22 
2.99 
2.84 
2.92 
3.16 
3.02 
2.91 



1.04 
0.92 
1.12 
1.11 



Table 8. — Frog VI, Kept at 22°, Killed after 8 Day.s 
Ash weight: 3.46 gm 



Weight of fresh tissue 



Weight of asli 



Per cent of labelled P 
administered found 
in 1 gm fresh tissue 



Right femur epiphysis 
Left femur epiphysis . 
Right femur diaphysis 
Left femur diaphysis . 
Right tibia epiphysis . 
Left tibia epiphysis . . 
Right tibia diaphysis . 
Left tibia diaphysis . . 

Right calf 

Left calf 



mgm 

150.6 

169.8 

398.9 

382.0 

263.9 

252.4 

304.3 

282.5 

gni 
1.520 
1.584 



Bones 
mgm 

37J 

37.6 
130.4 
122.3 

60.2 

60.7 
122.6 
120.9 

Muscles 



4.44 
2.66 
3.26 
3.20 
2.97 
3.12 
3.66 
3.42 



1.1 
1.2 



OIRCULATIOX OF PHOSPHORUS IX THE FROG 



391 



TaBlk 9. — Frog VII, Kki-t at 20 — 24% Kii.i.kd aftkii 12 Dav.s 

Weight: at tlu' start .">(]. 5 gm; at the end 49.5 gm 
Ash weight: 2.359 gm 



Weight of flesh tissue 



Kight femur epiphysis 
Left femnr epiphysis . 
Right femur diaphysis 
Left femur diaphysis . 

Right thigh 

Left thigh 

Right calf 

Left calf 



\Veiglit of ash 



i'er cent of luljelled 1' 
administered found 
iu 1 gm. fresh tissue 




than the replacement of phosphoitis in the bone tisstu\ From the tact 
that the ratio of the uptake of ^ap by 1 gm muscle tissue and 1 gm 
bone tissue decreases much with increasing temperature (see Table 10), 
we can conclude that the temperature coefficient of the penetration 



Table 10. — Comparison of the Labelled P Content 
OF Bones and Muscles 




I 

II 
III 
III 
IV 
IV 



V 

V 

VI 

VI 

VII 

VII 





Temperature: 2° 


12 




19.4 


12 




17.7 


21 




12.8 (epiphysis) 


21 




13.9 (diaphysis) 


22 




13.2 (epiphysis) 


22 




15.5 (diaphysis) 



Average 


value. . . 15.4 




Temperature: 22° 


8 




3.0 (epiphysis) 


8 




2.9 (diaphysis) 


8 




2.9 (epiphysis) 


8 




2.9 (diaphysis) 


12 




4.1 (epiphysis) 


12 




4.6 (diaphysis) 


Average vak 


le... 3.4 



392 ADVENTURES IX RADIOISOTOPE RESEARCH 

and subsequent incorporation of labelled phosphate into the organic 
compounds of the muscle of the frog is much greater than the tempera- 
ture coefficient of the formation of apatite crystals. 

EXCRETION OF LABELLED P 

In a few cases, we determined the percentage ^^P which was excreted 
by the kidneys of the frog. In one experiment, 1.5 cc. 0.6 per cent sodium 
chloride solution containing 0.008 mgm P as phosphate was injected 
into the lymph sack of the frog weighing 55 gm and kept at 18° . Urine 
was collected during 14 hours and the ^^P content of the urine was 
determined. It was found to make out 10.6 per cent of the ^-P injected 
while, in other experiments, 7.1 and 5.8 per cent, respectively, was 
found. 

UPTAKE BY THE FROG OF ^^p FROM A SOLUTION CONTAINING 

LABELLED SODIUM PHOSPHATE 

A frog weighing 88 gm was kept at 18° in 100 cc. 0.6 per cent sodium 
chloride solution containing 4 mgm labelled P as sodium phosphate. 
The solution was renewed every day. After the lapse of 21/2 days, the 
frog was washed, killed and its P content extracted. It was found to be 
695 mgm or 7.9 mgm per gram of fresh weight of the frog. The specific 
activity of this P was found to constitute 1/450 of the specific activity 
of the P of the solution in which the frog was kept. Thus, in the course 
of 2I/2 days, 1/450 of the total P of the frog was replaced by solution P. 
We investigated, furthermore, the activity of the inorganic P extracted 
from the liver of the frog which was found to show a specific activity 
amounting to 0.99 per cent of the specific activity of the solution P. 
It was, thus, 4.5 times more active than the average P of the frog. 



RATE OF RENEWAL OF THE PHOSPHORUS COMPOUNDS IN THE 

MUSCLE 

In the preceding sections, experiments were described in which the 
percentage of the administered ^sp present in the skeleton and the 
muscles was determined. In the following, we wish to discuss the rate 
at which the organic phosphorus compounds present in the muscles 
of the frog are renewed. We shall consider those cases of renewal^^ 

^^) It is conceivable that molecules are renewed whithout the spUtting off and 
reincorporation of phosphate group. 



CIRCULATION OF PHOSPHORUS IN THE FROG 39 IJ 

incorporated inio the organic molecules. For example, creatinephosphoric 
acid is degraded uikUm- splitting off of phosphate and resynthesized 
under uptake of phosphate radicals. If labelled phospha1<> ions arc prc- 
*?enl, they \vill have 1he same chance to be incorporated as have non- 
labelled ones. J.,et us assume 10*^ free phospha1(> ions pr(\s(>n1 in 1 he 
muscle cells to conlain 10 ^'^PO^ ions Avhiie from lo« P aloms isolated 
from hexosemonophosphale of the muscle tissue only 1 is ^'I*, 1hen we 
have to conclude that 10 per cent of the hexosemonophosphale^ mole- 
cules were renewed during the experiment under incorporation of fre(> 
phosphate. The ratios of the specific activities of the inorganic P and 
the organic P are, thus, a measure of the extent of renewal of the organic 
P compound which took place during the experimeni . When trying 
1o arrive at quantitative data we encounter the following difficulties: 
(a) The free phosphate extracted from the muscle tissue is partly cellular 
and partly extracellular phosphate; it is, however, the specific activity 
of the cellular phosphate only which is to be considered when calculating 
the rate of renewal, (b) The specific activity of cellular phosphate changes 
during the experiment, the change being due, for example, to an increas- 
ing influx of labelled phosphate into the muscle cells. In this connection 
it should also be mentioned that the method permits to distinguish 
between renewed and non-renewed, between "old" and ''new" molecules,- 
but no information is supplied on the point whether the molecules are 
repeatedly renewed in the course of the experiment or not. 

As to point (a), to account for the share of the extracellular phosphate 
in the total phosphate of the muscle tissue, we must know the specific 
activity of the plasma phosphate which we assume to be identical with 
the specific activity of the extracellular phosphate. We must also know 
the phosphate content of the plasma and that of the muscle tissue and, 
finally, the size of the extracellular space. The last mentioned magni- 
tude can be determined in each case by administering simultaneously 
with the labelled phosphate labelled soclium^^), or it can be assumed that 
the extracellular space makes out 14 per cent of the weight of the muscles. 
Another procedure Avhich mc used repeatedly is the following. We remove 
one leg of the frog 1 hour after the start of the experiment and determine 
the specific activity of the free muscle phosphate P. After further 3 hours, 
we extract the phosphate of the other gastrocnemius and determine the 
specific activity of the free phosphate P. If, within 1 hour, a proportional 
partition of ^^p between plasma phosphate and the extracellular phos- 
phate took place, then the increment of the specific activity of the tissue 
phosphate between 1 hour and 4 hours is solely due to an increase in 
the specific activity of the cellular inorganic P. By this method, we can 
determine the percentage of cellular P which was replaccnl in \hr muscle 



fi) 



Comp. G. Heve.sy and (). Rebbe, Acta Physiol. Scand. 2, 171 (1940). 



394 ADVENTURES IN RADIOISOTOPE RESEARCH 

by plasma P between 1 hour and 4 hours after the start of the experi- 
ment. 

The fact that the inorganic P of the tissue is partly of extracellular 
origin will lead to an overestimation of the activity of the cellular inor- 
ganic P and, thus, to an underestimation of the renewal figures of the 
organic P compounds. This source of error is mainly to be considered 
in experiments of short duration carried out at low temperature. On 
the other hand, even if the greatest precautions are observed, we risk 
a decomposition of some of the creatine phosphate present in the tissue 
prior to the separation of the inorganic P. Such a decomposition will 
lead to a decrease in the specific activity of the inorganic P, the inor- 
ganic P originating from creatine P being on the wdiole less active than 
the "free" phosphate P. We shall, thus, underestimate the specific 
activity of the inorganic P and, correspondingly, overestimate the rate 
of renewal of the organic P compounds. This error will also be larger 
in experiments of short duration carried out at low temperature. We 
wish to mention a further possible experimental error. If the free phos- 
phate is not precipitated quantitatively, we risk to find some strongly 
active phosphate in the creatine phosphate fraction. A non-negligible 
amount of phosphate may remain in solution in cases in which the 
amount of P to be precipitated is very small. 

The following objection can be put forward regarding the calculation 
of renewal rates from the ratio of the specific activities of the inorganic 
P and the P split off from organic compounds. The P secured as inorganic 
phosphate might even after the most careful handling of the tissue 
have been largely present not as free phosphate in the tissue cells but 
incorporated in very labile compounds which were decomposed in the 
course of the extraction process. It is possible that this is the case, it 
is even quite possible that a large part of the inorganic P extracted 
as such from the muscle cells was originally present incorporated in very 
labile compounds and was decomposed during the extraction process. 
General experience indicates, however, that very labile phosphorus 
compounds are renewed at a fast rate and we can, therefore, expect 
the P of such labile phosphorus compounds to obtain within a short 
time a similar specific activity as shown by the inorganic P present 
in the cells. Should that not be the case, then the comparison of the 
specific activity of the "inorganic" P with that of the P split off from 
the organic compound in question, would obviously lead to an overesti- 
mation of the rate of renewal. 

The specific activity of different P fractions is seen in Tables 11 — 18. 

Though all precautions were taken to prevent decomposition of 
creatinephosphoric acid it is difficult to state whether the variations in 
the values obtained for the rate of renewal of creatinephosphoric acid 
molecules in some of the experiments are genuine or are due to a more 



CTRCrLATIOX OF L'HOSI'lIORUS IX THK KK()(i 



395 



or less successful prevention of the decomposition of 1li(» creatinephos- 
phoric acid prior to the removal of the inorganic phosphale of Uio muscle 
1 issue. 

The resvills c^f an (experiment, in whicli the frog was kept at 2(f for 

Table 11. — Spjxific Activity of Phosphor is Fractions Extracted from the 
Gastrocnemius of a Frog, 4 Hours after Injecting Labelled Sodixxm Phos- 
phate INTO THE Ly.MPH SACK. TeMP. : 2° 



Fnictiou 



1' content 
in mgm 



Aotivitj- ill per Per cent of tot^l 
cent of tlie Stan- activity adminis- 
dard preparation tered per mgm P 



Holative 
specific 
activity 



I. Inorganic P 


0.313 


69.5 


0.SS8 


100 


II. Inorganic -)- creatine P 


0.035 


100 


0.428 


48.2 


III. Creatine P calculated as II — I 


0.622 


30.5 


0.20 


22.1 


IV. Creatine P (isolated) 


0.362 


17.1 


0.1!) 


21.4 


V. Pyrophosphate -f- hexose P ... 


0.215 


8.3 


0.16 


17.4 


\'I. Acid soluble residual P 


0.113 


3.2 


(J. 11 


12.7 


VII. Non acid soluble P 


0.500 


1.6 


0.013 


1.4 



We denote as pyrophosphate + hexose P the inorganic phosphorus obtained after the hydrolysis of a 
fraction for one hour at 100" in 1 X. HjSOj after tlie removal of the inorganic and creatine P. 

4 hours and then for 1 hour at 0°, is seen in Table 18. The muscles were 
immersed in liquid air and treated with cold 5 per cent trichloracetic 
acid. The extract was sucked through a glass filter into cooled Fiske's 
leagent. These operations took 2 minutes. In this experiment, we tested 

Table 12. — Specific Activity of Phosphortts Frac- 
tions Extracted from the Gastrocnemius 
OF frogs. Temp.: 2° 



Time of the 
experiment hours 



Fraction 



Specific activity 



4 
4 
4 
4 
3 
3 



Inorganic P 
Creatine P . 
Inorganic P 
Creatine P . 
Inorganic P 
Creatine P . 



100 

14.8 
100 

21.4 
100 
8.8 



to what extent the inorganic P is precipitated by Fiske's reagent. 
After precipitation of the "free" phosphate, 60 mgm sodium phosphate 
were dissolved in the filtrate, the phosphate was then precipitated and 
its activity tested. If the first precipitation was strictly quantitative, 
this second precipitate should be inactive. The counter registered 228 
counts while, in the case of the first precipitate. 2500 counts were regis- 



396 



ADVENTURES IN RADIOISOTOPE RESEARCH 



tered. When the 228 non-precipitated counts are considered, the specific 
activity of the creatine P fraction works out to be 14.1 instead of 15.6. 

The same technique was used in the following experiments. 

The lowest value found for the percentage renewal of creatinephos- 
phoric acid molecules in the course of 4 hours at 0° is 9 and in the course 
of 17.5 hours 10 while, in most experiments, appreciably larger figures 
were found. The rate of renewal of the creatinephosphoric acid molecules 

Table 13. — Specific Activity of Phosphorus Frac- 
tions Extracted from the Gastrocnemius of a Frog 
40 Hours after Injecting Labelled Sodium 
Phosphate. Temp.: 2° 



Fraction 



JBelative specific 
activity 



I. Inorganic P 

II. Inorganic -{- creatine P 

III. Creatine P (calculated a.s II — I) 

IV. Creatine P (determined) 

V. Pyrophosphate -f- hexose P ... 

VI. Acid soluble residual P 

VII. Non acid soluble P 



100 
(32.7 
33.7 
34.4 
22.8 
11.3 
2.1 



Table 14. — Specific Activity of Phosphorus Frac- 
tions Extracted from the Gastrocnemius of a frog 
24 Hours after Injecting Labelled Sodium 
Phosphate. Temp.: 20° 



Fraction 



Relative specific 
activity 



Inorganic P 

Creatine P 

P hydrolysed in the course of 1 lionr 
Acid soluble residual P 



100 
9.5 
91 
36 



Table 15. — Specific Activity of Phosphorus Frac- 
tions Extracted from the Gastrocnemius of a Frog 
400 Hours after In.jecting Labelled Sodium 
Phosphate. Temp.: 20° 



Fraction 



Inorganic P 

Creatine P 

P hydrolysed in the course of 1 hour 
Non acid soluble P 



Relative specific 
activity 



100 
99 

87 
16 



CIRCULATION- OF I'lrOSPHORUS IX THE FllO(; 



397 



Table 16. — Specific Activity of thk Phosphortts Fractions of thk Musclks 
OF A Frog Kept at 20° for 4 Hours and Subseqentta- at 0° for 1 ITot:r 



Fniction 



r content 
ill nitrin 



Counts 
por min. 



Specific 
nctivitv 



Relative 
specitic 
iictivitv 



Inorganic P 
Creatine P . 
Hexose P . 



Left gastrocnemius 



0.055 
0.240 
0.143 



427 

290 

98 



7770 

1210 

()8ti 



100 
15.() 

8.7 



Inorganic P 

Creatine P 

Ilesidne after 17 hours livdrolysis 



Right gastrocnemius -{- .sartoriu.s 
0.350 j 2500 7150 | 100 

0.908 I 1010 I 1110 ].-..() 

0.372 i 45 I 120 1.(58 



of the resting frog is, thus, quite appreciable even at 0° though not as 
high as stated in a preHminary note'^^ At 20° the lowest figure found 
after 4 hours is 16 per cent. 



Rate of interaction of the plasma phosphate and the cellular phosphate 
of the muscle tissue 

In the preceding section, we calculated the rate of renewal, of the 
organic P compounds present in the muscle tissue by comparing the 
^-P content of the tissue inorganic P with the ^^P content of the phos- 

Taule 17. — Specific Activity of the Phosphorus Fractions of the MtrscuEs 

of 2 Frogs Kept .^.t 0° for 17.5 Hours 



Frosj 



Friictiou 



P content 



Specific 
activity 



Gastrocnemius 



Sartorius 



II 



Inorganic P 

Creatine P 

Product of 100 min hydrolysis . . . 
Residue after 120 hours hvdrolvsis 



Inorganic P 

Product of 7 mill liydrolysis . . 
Product of 17 hours hyth'olysis 
Residue 



j Inorganic P 



Gastrocnemius i Creatine P 



II ( Inorganic P 

Sartorius |: Creatine P . 



0.330 
0.474 
0.220 
0.070 

0.71)'.t 
I.05(i 
0.75S 
0.24() 

0.39S 
0.452 

0.475 
0.r)!t4 



100 

29. S 

27.0 

2.0 

100 
25.1 
19.5 
(5.8 

100 
II.O 

100 
10.1 



^i^G. IIevesy and O. Rebbe, Xalure 141, 1097 (1938) 



398 



ADVENTURES IN RADIOISOTOPE RESEARCH 



Table 18. — Specific Activity or Phosphorus Frac- 
tions Isolated prom Different Organs of a Frog. 
AFTER Administration of Labelled Phosphate 
During 45 Hours at 20° 



Fraction 



Specific aotiTity 



Plasma P 

Corpuscle P 

Gastrocnemius inorganic P 

Gastrocnemius creatine -\- pyrophosphate P 

Liver P 

Epiphysis P 

Diaphysis P 



100 
3.6 
4.9 
5.3 
10.1 
0.35 
0.20 



Table 19. — Specific Activity of Phosphorus Frac- 
tions Isolated from Different Organs of a Frog 
AFTER Administration of Labelled Phosphate 
During 4 Days at 22° 



Fraction 


Specific activity 


Plasma P 


100 


Gastrocnemius inorganic P 

Gastrocnemius creatine P 

Sartorius total acid soluble P 

Gastrocnemius phosphatide P 


8.3 
7.4 

8.5 
1.5 



phorus extracted from the compound in question. In the following, 
we shall discuss the interaction of the plasma phosphate with the cellu- 
lar phosphate. This is clearly a very different problem, the rate of inter- 
action between the plasma phosphate and the cellular phosphate being 
determined by the permeability of the cell membrane. 

The low rate at which phosphate ions migrate through the membrane 
of the cells of the gastrocnemius is seen in Tables 18 and 19. In the course 
of 4 days at 22° only somewhat less than 1/10 of the P atoms present in 
the labile P compounds got replaced by plasma P. The molecules of the 
labile P compounds were repeatedly renewed during this interval and 
many P atoms present in the muscle cells interchanged lively; however, 
the interchange between cellular and extracellular P took only place 
on a restricted scale. 

The results of further experiments in which the activity of the plasma 
was compared with the activity of the muscle is seen in Table 20. 

To keep the plasma activity at an approximately constant level 
throughout the experiment, 0.4 cc. solution was injected at the start 
of the experiment and further 0.08 cc. every hour. As seen in Table 20, 
within 1 hour and 4 hours the activity of the plasma changes only 



CIKC'LLATION OF PHOSPHORIS IX THE KllOG 



399 



slightly, the average being 108, taking the end value to be 100. The 
values obtained for the specific activity of the tissue P are seen in Table 
20 and Fig. 3. 

In some eases, very low values were obtained for the distribution 
ratio of IuIjcHchI phosphate between plasma and muscle tissue. The fact 



200- 



150 - 






o 
o 



o 100- 



u 

•> 
a. 



o 
o 



50- 




Gasrrocnemius 



Tibia epiphysis 
Tibio diaphysis 



Time in hours 
Fig. 3. Specific activity of tissue P. 



that in these experiments frogs kept through the winter were used, the 
experiment being carried out in the spring, suggests the explanation 
that poor circulation may be responsible for the low values obtained. 



Table 20. — Activity of Different Fractions of the Frog 1 Hour and 4 Hours, 
Respectively, after the start of the Experiment 

Temp.: 22° 



Time 



1 hour 



Fraction 



4 hours 



Plasma 

Gastrocnemius 
Epiphysis .... 
Diaphysis .... 



Plasma 

Gastrocnemius 
Epiphysis .... 
Diaphysis .... 



Fresh weight 
in mf'm 



P content 
in mgm 



Activity 

per mgm 

fresh weight 



98.7 

861.8 

94.1 

67.6 

1267.5 

812.4 

38.8 

60.1 



0.00344 
1.3.5.5 
6.100 
6.830 

0.044 
1.300 
3.61 
6.410 



116(1) 
32.7 
224 
235 

100 

93.5 
755 

578 



Specific 

activity 

of P 

116 
0.72 
0.12 
0.08 

100 
2.03 
0.28 
0.19 



<•> Taking the 4 hours value to be 100. 



400 



ADVENTURES IN RADIOISOTOPE RESEARCH 



Increment in the Specific Activitie.s between 
1 HorR AND 4 Horns 



Fraction 


Experimental 
value 


Value corrected 
for the change 
in the specific 
activity of the 
plasma 


Gastrocnemius 


1.34 
O.K) 
0.11 


1.21 

14S 


Dianlivsis 


0.102 







^^P content of the liver fractions 

As in the case of mammalia, in the frog the liver phosphate interacts 
at a much faster rate with the plasma phosphate than does the muscle 
phosphate. The fast rate of renewal of the acid soluble P compounds 

Table 21. — Specific Activity of the Phosphorts 

Fractions Extracted from the Organs of the Frog 

Kept at 15° and the Organs of the Rabbit 10 Hoirs 

after Administration of Labelled Phosphate 



Fraction 



Specific activity 



Frog 



Rabbit 



Plasma 

Gastrocnemius inorganic P 

Gastrocnemius total acid soluble P 

Liver inorganic P 

Liver pyrophosphate P , 

Liver hexosemonophosphate P . . . . - 

Liver residual acid soluble P , 

Liver phosphatide P 



100 


100 


2.11 


15.5 


1.49 


11.0 


12.9 


85 


15.2 


— • 


8.9 


— 


3.5 


— 


0.04 


12.8 



Table 22. — Distribution of ^^p between 
Plasma and Corpuscles of the Frog 



Time of experi- 
ment hours 




Distribution ratio of ^-1' 
between corpuscle and 
plasma of equal weight 



10 


15° 


0.28 


14 


20° 


1.1 


45 


20° 


3.6 



and the very slow renewal of the phosphatides of the liver of the frog 
are seen in Table 21. This table contains also corresponding data for the 
P fractions of the rabbit. 



CIRCULATIOX OF PHOsPHOliUS I.\ THE HlOG 401 

3-P content of the red corpuscles 

As seen in Table 22, a very slow interaction was found to take place 
l)etween the plasma P and the corpuscle P present in the nucleated 
corpuscles of the frog. 



Summary 

The rate of absorption of phosphate injected into the lymph saek of the frog 
was stviclied using radiophospliorus as an indicator. The niaxinuini amount of 
labelled phosphate present in the circulation after subcutaneous injcMjtion at any 
moment was found to be 3 to 4 per cent of the amount administered, thus a 
similar value as fountl in the case of mammalia. 

While at 2° after the lapse of 1 to 3 weeks 1 gin bone tissue contained about 
lo times as many labelled P atoms as 1 gm muscle tissue, the corresponding 
ratio was found to be but 3 at 22°, showing that the temperature coefficient of 
the penetration of labelled phosphate into the muscle cells followed by incorpora- 
tion of labelled P into the phosphorus compounds of the muscle tissue is much 
larger than the temperature coefficient of the formation of labelled bone apatite 
crystals. 

The amount of labelled phosphate excreted by the kidneys and the amount 
of labelled phosphate taken up by the frog kept in physiological sodium chloride 
solution containing labelled phosphate were investigated. 

The rate of renewal of various acid soluble P compounds extracted fi-om the 
gastrocnemius of the frog was determined b\' comparing the specific activity 
of the inorganic P extracted from the muscle with the specific activity of the 
phosphorus split off from various organic compounds of the muscle tissue. Creatine- 
phosphoric! acid molecules, adenosintriphosphoric acid molecules, and also hexose- 
monophosphate molecules were found to be renewed at an appreciable rate even 
at 0". The rate of renewal was found to increase with decreasing chemical stabihty 
of the compound and with increasing temperature. 

The rate of interaction of the plasma phosphate with the phosphate of the 
mviscle cells was found to be very much lower than the rate of interaction of the 
free cellular phosphate with the phosphate of .several organic phosphorus com- 
pounds. 

The rate of penetration of labelled phosphate into the liver celLs is much faster 
than the rate of penetration into the muscle cells. The rate of interchange of plasma 
phosphate and the phosphate of the corpuscles was found to be fairly low. 



2G Hevesv 



402 ADVENTURES IN RADIOISOTOPE RESEARCH 



Comments on papers 36 — 38 

In oo operation with Parnas (1938) in in vitro experiments, the incorporation of 
the phosphate group into various acid-soluble phosphoiiis compounds was studied. 
It was found that a phosphate group may be transferred fiom one organic molecule 
to another without passing the inorganic stage. For example, when glucose-1 
phosphoric acid (Cori ester) is transformed in the presence of muscle extract and 
labelled inorganic phosphate into glucose- 6-phosphoric acid (Robison ester) the 
esters do not become labelled. 

Simultaniously Meyerhof, et al. studied the rate of interchange between 
inorganic and pyrophosphate P in muscle-extract and found that after the lapse 
of 20 sec, a 47 per cent interchange took already place. 

The first in vivo studies on the turnover of acid-soluble phosphorus compounds 
of the muscle tissue were carried out with frogs (paper 36). The turnover rate 
of the acid-soluble constituents was found to be influenced to a much higher 
degree by temperature- changes than by the foimation of the labelled bone apatite 
crystals. When investigating the rate of renewal of the acid-soluble organic phos- 
phorus compounds of the rabbit (paper 37), the inorganic phosphate content of the 
plasma was kept at an almost constant level throughout the experiment, which 
took 50 days. This made it possible to arrive, by comparison of the specific activ- 
ities of the cellular inorganic P and the organic P at the end of the experiment, 
at a renewal figuie of the latter. In experiments of 50 days duration, the results 
can be expected not to be influenced much by the participation of slowlj^-formed 
intermediary products, or the specific activity of extracellular phosphate much 
to differ from that of the intracellular one, in contrast with experiments taking 
a few hours only. 

Recently in ^^O of Hg^^O a very suitable indicator in the study of the turnover 
of phosphorus compounds of the muscle cells was found by Fleckenstein et al. 



References 

G. Hevesy, T. Baranowski, A. J. Gutke, P. Ostern and J. K. Parnas (1938) 

Acta Biol. Exp. 12, 34. 
O. Meyerhof, P. Ohlmeyer, W. Gentner and H. Maier-Leibnitz (1938) 

Biochem. Z. 298, 396. 
J. Sachs, Isotonic Tracer in Biochemistry and Physiology , New York, 1953. 

A. Fleckenstein, E. Gerlach, I. Janke and P. Marmier, Z. Physiol. C/iem.( 1960) 
271, 75. 



Originally published in Experientia 7, 144 (H).51) 

39. TURNOVER RATE OF THE FATTY ACIDS OF 

THE LIVER 

G. Hevesy, R. Ruyssen and M. L. Beckmans 

From the Pharmaceutical Institute of the University of Gent and Institute 

for Research in Organic Chemistry, Stockholm 

The rate of renewal of the fatty acids of the hver and other organs has 
been repeatedly determined by making use ol' deuterium, carbon 
13 and carbon 14 as an indicator. In early experiments Schoenheimer 
and RiTTENBERG^i) administered heavy water to mice and kept the 
deuterium content of body fluids at a constant level throughout the 
experiment. The saturated fatty acids of the mouse reached half of their 
maximal deuterium content in the time of 5 to 9 days. The deuterium 
content of the saturated acids was higher than that of the unsaturated. 

Bernhard and Schoenheimer<2> isolated the fatty acids of the 
intestinal wall, the kidneys and the liver of the mouse and found the 
saturated fatty acids to have an appreciably higher deuterium^^) content 
than had that of the unsaturated ones. They estimate the half-life time 
of the average saturated fatty acid molecule in the liver of the mouse 
to be al)out 1 day, while the half-life time of the total fatty acids in the 
rat liver was found by Stetten and Boxer^^) to be 1.9 days. 

The finding of Rittenberg and Bloch<^> that the feeding to mice of 
acetate containing ^^C " in the carboxyl group leads to the formation of 
fatty acids containing ^^Q, opened the way to the application of i^C 
and i^C in the study of the rate of formation of fatty acids. They found 
a more rapid incorporation of ^^C in to the saturated than into the 
fatty acids. The i^C concentration of the carboxyl carbon atoms of the 
saturated fatty acids was approximately twice as high as the average of 
all the carbon in the saturated fatty acids. The most plausible distribu- 
tion which will explain these data is one in which the labelled carbon is 

(1^ R. Schoenheimer and D. Rittenberg, J. Biol. Chem. 114, 381 (lOSG). 
D. Rittenberg and R. Schoenheimer, J. Biol. Chem. 114, 381 (1936). 

(-^ K. Bernhard and R. Schoenheimer, J. Biol. Chem. 133, 713 (1940). 

(3) K. Bernhard and F. Bullet, Helv. chim. Acta 26, 75, 1185 (1943). 

(*)D. Stetten Jr., and G. E. Boxer, J. Biol. Chem. 155, 231 (1944). 

(5)D. RiTTENBERGand K. Bloch, J. Biol. Chem. 154, 311 (1944). K. Bloch 
and D. Rittenberg, J. Biol. Chem. 159, 45 (1945). D. Rittenberg and K. Bloch, 
J. Biol. Chem. 160, 417 (1945). 



26* 



404 



ADVEXTURES IX EADIOISOTOl'E RESEARCH 



present at every other carbon atom; i.e. at the odd number carbon 
atoms of the fatty acids. Later work^^^ showed that at least 25 percent 
of the carbon atoms of the fatty acids are derived from acetate. Evidence 
was also obtained that acetic acid can furnish every carbon atom of the 
molecule. 

Recently the rate of turnover of fatty acids has been re-investigated 
with the aid of acetic acid labelled by ^HJ in the carboxyl group by Pihl 
et alM^ The percentage renewal of the fatty acid molecules is determined by 



o 
O 
u 

u 
a> 
a 



~" 














' 










— 


























— 




i 


) 










































1 — 




AOO - 


fi 


)\ 


















































1 


i 


















































1 


















































IV 


1 
















































500- 














































— 




w 


1 
















































\ 




n 




































1 










k " 


r * 


K 






































'^uu- 


\ 


V 




S 


t 






















— 1 




















h 


f 

5 


V 






V 










































^ 


\ 






> 


k 




































: / 


A 




^ 


V 


»^ 


s^ 






b 
































•lOU 


■ V 


( 






\ 


bC 


=-^ 


r 


li 


*— 


— 


'_* 


li 


pi 


















ir 












'■*. 


























^ 


* * 


"■ 


:u 








^ 


























































































_ 




□ 









10 20 50 40 SO 80 100 120 



180 



21*0 
Time,minuhes 



Fig. 1 . Change of the specific activity of tlie liver fatty acids with t ime. 



comparing the ^^C content of the fatty acid carbon at the end of lhe 
experiment with the average value of the ^^C content of the precursor 
carbon which prevailed during the experiment. To arrive at the last 
mentioned data, phenyl-DL-aminobutyric acid was fed simultaneously 
with labelled acetate to adult rats kept on fat free diet, and consecutive 
samples of the excreted acetyl derivatives were analysed.^^^ Though the 
labelled acetate content of the diet was kept constant, the isotope con- 
centration of the acetyl group was found to increase in the course of the 
30 days period with about 30% of the initial value. This increase was 
shown to be due to the catabolism of the labelled higher fatty acids 
I'ormed during the experiment. The metabolic products of the labelled 



^-' K. PoNTECORVO, D. RiTTENBERG and K. Block, J. Biol. Cheiu. 179, 893 
( 1949). A. Pihl, K. Bloch and H. S. Anker, J. Biol. Chein. 183, 441 (1950). 

(2^ K. PoNTECORVO, D. RiTTENBERG and K. Bloch, J. Biol. Chem. 179, 893 
(1949). 

'^Uv. Bloch and D. Rittenberg, J. Biol. Chem. 159, 45 (1945). 



■rrRXOVEE 15AIK OF T1{E FATTV AflDS OF 1111; IIVER 405 

fatty acids contiihiilo in 1hrs(> long-time expoiimonts significant quanti- 
ties of labelled acetyl groups 1o the acelic acid pool which supplies i^C 
to the newly formed fatty acid molecules. 

Th(> saturated I'atty acids of liver were found to reach half of their 
maximal isotope concentration in less than 1 day, Ihe unsaturated acids 
in al)0ul 2 days. Much longer time is necessary to reach a corresponding 
i^C concentration in the fatty acids of the carcass, 16 — 17 days for 
saturated and 19 — 20 days for the unsaturated acids. This difference 
was also shown in recent work of Popjak and Beeckmans(^). 

In an investigation on the effect of changes in the metabolic rate on 
the incorporation of ^'*C into tissue fractions, we determined the specific 
activity of the liver fatty acids of the mouse following injection of 
carboxyl labeled acetate in experiments of 10 to 180 min duration. The 
results obtained, which are discussed in this note, suggest, the existence 
of a fatty acid fraction in the mouse liver of much shorter half-life than 
about 1 day. which was found in the various experiments mentioned 
above. 



EXPERIMENTAL 

In each experiment 45 to 72 mice were injected intraperitoneally 
with 0.2 ml of 0.8% sodium chloride solution containing 2 — 4 microcurie 
(0.2—0.4 mgm) of sodium acetate labelled in the carboxyl group. The 
animals were divided in 5 — 6 equal groups and killed after 5 to 240 
min. 

The pooled organs were frozen in solid COg. The ground tissue was then 
extracted for 3 hr with a boiling mixture of ether-alcohol 1:3. The filtrate 
obtained was evaporated in vacuo and the residue extracted with pet- 
roleum-ether. The residue obtained after evaporation of the petroleum- 
ether was saponified for 8 hr with 10 ml of 40% KOH solution and 20 
ml of alcohol on a boiling waterbath. 

The solution was extracted three times with petroleum-ether in order 
to remove the insaponifiable matter. The aqueous solution was then 
neutralised with 40% HoSO^ solution and extracted three times with 
petroleum-ether. 

The petroleum-ether solution on evaporation gave the fatty acids. 
The determination of the radioactivity was carried out with a Geiger 
counter, 8 mgm of each sample on an aluminium disk of 5 mm diameter. 



(i^G. Popjak and .M. L. liEEOKMANS, Biochem. J. 69, o47 (19.50). 



406 ADVENTUKES IN RADIOISOTOPE RESEARCH 

DISCUSSION 

The relative specific activity of the total fatty acids extracted from 
the liver of mice killed at different times after intraperitoneal injection 
of acetate labelled in the carboxyl group is plotted in Fig. 1. In view 
of the fact that the five experiments, the results of which are recorded, 
were carried out with different strains of mice, we plotted the results 
of each experiment by taking the value obtained after 60 min experi- 
ment to be 100. Each point indicates a value obtained by extracting 
the fatty acids of to 12 pooled livers. The total number of mice involved 
in these experiments amounted to 250. 1 mgm of fatty acid of the liver of 
a 20 gm mouse contains 8.2 X 10^^ p^rt of the ^^0 administered. This figure 
indicate thes value corresponding to the highest peak of the curves. 

It takes several minutes until the injected labelled acetate or its 
decomposition products penetrate into the liver and are incorporated 
into fatty acid molecules. Correspondingly, the specific activity of the 
fatty acids increases for the first minutes. This time was determined 
for the rat to be 15 min. <^) The increase in the specific activity of the 
fatty acid precursors with time is soon followed by a decrease. While 
the endogenous inactive acetate is constantly being produced, about 1"2 
mgm are formed daily per 100 mgm of rat tissue^-), the injected labelled 
acetate is not replaced. ( -orrespondingl}^ after a while fatty acid mole- 
cules w^ill be synthesized in the liver from almost inactive precursors. 
The time will also arrive when the labelled fatty acid molecules present 
will be renewed a second time from less active precursors than the first 
time. Active fatty acid molecules will thus be replaced by less active 
ones, and the specific activity of the average fatty acid molecule shall 
now decrease with time. The rate of decrease will be determined by 
the half-life time of the liver fatty acid molecules. Let us assume that 
after the lapse of 30 min. no further active fatty acid molecules are 
formed in the liver, the precursors being no longer active, and corre- 
spondingly the specific activity of the fatty acid of the liver Avill decrease 
with time according to the formula 

In 2 t 

S^ = 8,e- ^~ 

where Sq denotes the specific activity after 30 min., S that at any later 
time t, while T = half-life time of the fatty acid molecules in the liver. 
Assuming T — \ day as found by different workers in feeding experi- 
ments, than in the interval between 30 and 6<) min. the decline in the 

^1^ R.G.Gould, F. M. Sinex, I. N. Rosenberg, A. K. Solomon and A. B. 
Hastings, J. Biol. Chem. 177, 295 (1949). 

(2) K. Block and D. Rittenber(;, J. Biol. Chem., 159, 45 (1945); A. Pihl 
K. Block and H. S. Anker, J. Biol. Chem. 183, 441 (1950). 



TURNOVER RATE OF THE FATTY ACIDS OF THE LIVER 



40: 



specific activity should bo al)()ut 3% only. Wo assumed in \\w above 
calculation that all formation of lal)olled fatty acid molecules ceases 
after the lapse of 30 min. As this assumption does not hold strictly, the 
decline in the specific activity of fatty acids in the interval between 30 
and 60 min. is even less than 3%. 

The data of Fig. 1 indicate a very much larger decrease in the specific 
activity of the fatty acids than 3% in the interval between 30 and 60 





















































4— 
> 




/ 


. 








-- 


— , 





. _ _ 





-4- 


— 


— 


— 


— 


— 
















o 

u 


i 










' 






















A 












r ' 


n 


1 
1 


n 


1 ^- 


^ " 

"^1 


fn 


-*^ 


• • • • 


• « • •. 


p.... 


•• C 


p 




























> 




M 


•> 


< 


) 










































o 

01 


W 


h 


















































L_ 



















































40 



60 



80 



100 



120 



180 



240 

Time, minures 



Fig. 2. Change of the specific activity of the brain fatty acids with 

time. 



min after administration of the labelled acetate. The decrease amounts 
to about 50%. These results suggest the presence of a rapidly renewable 
fatty acid fraction in the liver of the mouse. 

In experiments in which the labelled acetate is fed for days and the 
specific activity of the fatty acids in the liver daily determined, the 
presence of a minor fraction of rapid renewal rate cannot be expected 
to be observed. 

We investigated also the change of the specific activity of the brain 
fatty acids and muscle fatty acids with time. The specific activity of the 
brain fatty acids was found, as seen in Fig. 2, to increase rapidly with 
time in the course of the first minutes and then to remain almost constant. 
We lack thus any indication of the presence of rapidly renewed fatty 
acid faction in the brain. The specific activity values of the muscle 
fatty acids extracted from different groups of mice fluctuate consi- 
florably. An indication of a decrease of the specific activity figures 
within a 240 min. observation period is, however, also in this case absent. 



Summary 

Following the injection of in its carboxyl group labelled acetate to mice, the 
specific activity of the fatty acids extractes from the liver after the lapse of 30 
minutes is found to be appreciably lower than measured a few minutes after injec- 
tion. It follows from this finding that the fatty acids of the liver contains a rapidly 
renewable fraction. 



Originally communioatod ii Arch. int. pharmacodyn . 86, 33 (1951) 

EFFECTS OF DINITRO-CYCLO-PENTILPHENOL ON THE 
INCORPORATION OF LABELLED ACETATE CARBON (^ C) 

INTO TISSUE FRACTIONS 

M. L. Beeckmans, H. Casier and G. Hevesy 

Institute for Research in Organic Chemistry, University of Stockholm and 
J. F. Heymans Institute, Department of Pharmacology. University of Ghent 

As found in studies in which labelled acetate (^^(') was used as an indi- 
cator, acetate is rapidly metabolized in the animal (1) body. While 
the bulk of the administered acetate carbon is soon exhaled, some is 
incorporated into tissue fractions. 

Not only does the amount of administered labelled acetate diminisii 
rapidly with time, but the specific activity of the body acetate dimi- 
nishes as well. This is due to the constant new formation of endogenous 
non active acetate, which is, at least in the early phases of the experi- 
ment, practically inactive. 

As an effect of the diminution of the specific activity of the body 
acetate, the sensitivity of the radioactive indicator strongly increases 
with time. One count of acetate ^^C which indicates the presence of 1 
microgram of acetate or degradation product of acetate, at an early 
phase of the experiment may indicate the presence of 10 micrograms at a 
later phase. 

A change in the metabolic rate of acetate or its degradation products 
reflects itself in a change in their specific activity. Correspondingly a 
change in the metabolic rate will lead to a change in the ^^C content of 
tissue fractions and by following these changes we may conclude if 
and to what extent metabolic changes took place in the organ considered. 
If the CO2 production is due to metabolic interference slowed down from 
90 p.c. to 89 p. c, the incorporation of '^K' into tissue fractions may 
increase from 10 to 11 °'(, thus with as much as 1/10. 

Gould and assoc. (2) in experiments with adult rats, demonstrated 
the rapid disappearance of the injected labelled acetate carbon through 
exhalation as C'Og. The cumulative exhalation amounted in 4 hours 
to 87 % of the amount injected. After the lapse of 30 min about 1/3 
was still present in organic form in the rat, 1/3 in inorganic form, while 
1/3 is excreted as COg. The administration of labelled acetate does not 
interfere with the normal metabolic processes. Acetate is constantly 
produced and metabolized in the animal organism, the daily production 



INCORPORATION OF LAB. ACETATE CARBON ("C) INTO TISSUE FRACTIONS 409^ 

of acetate in a 100 gm rat amounting according to Block and Ritten- 
BERGandPiHLe^a/. (1, 3) to 15-20 mM. 

The effects of X-rays on animal tissue was found, in experiments in 
which labelled acetate was administered to mice, to influence markedly 
acetate metabolism (4). This observation induced the investigation of 
Ihe effects of metabolic inhibitors and accelerators on acetate meta- 
bolism. 

Previous work with mice carried out by Ruyssen, Beeckmans and 
one of us (5) showed an increased incorporation of ^^C in the urethano 
injected animals. 

In the present investigation IIk^ effect of a metabolic ac^celerator, that 
of dinitro-cyclo-pentylphenol (D.P.P.) was studied on ^^C incorporation 
into the total tissue and the total fat of various organs of the mouse. We 
choose dinitro-cyclo-pentylphenol as IIeymans and Casier (6) found 
this compound to stimulate cellular metabolism to a larger extent than 
any other dinitro-compound studied. 

Dinitro-cyclo-pentylphenol being a metabolic accelerator its effect 
on cellular metabolism can be expected to reflect itself in the rate of 
i^C incorporation into tissue fractions. 

As shown by different authors, oxygen consumption is increased 
after administration of 2.4-dinitrophenol and similar compounds. 

Terada and Tainer (7) injected subcutaneously 20 mgm of 2—4 dini- 
trophenol perkgm to 33 gm. rats and found an increase of about 25% 
in the oxygen consumption 20 min after injection. After the lapse of 
2 hours, normal oxygen consumption was again observed. In the case 
of adult rats the same effect could be obtained by injection of 15 mgm 
per kgm body weight. 

A slight stimulation of oxygen consumption was observed by Loomis 
and LiPMANN (8) in their experiments in which the effect of small amounts 
of dinitro-phenol on kidney homogenate metabolism was studied and a 
marked lowering of phosphorylation observed. The inhibition of coup- 
ling between oxidative processes and the formation of high energy bonds 
was found by these authors to be a conspicuous effect of dinitro-phenol. 



EXPERIMENTS 

Five groups of five adult mice each, were injected with 0.3 cc. of a 
solution containing 0.16% of sodium dinitro-cyclo-pentylphenol . (20 
mgm for 1 kgm of animal). 

Five groups of controls were injected at the same time with 0.3 cc. 
of a physiological sodium-chloride solution. 

Ten minuten afterwards all groups were injected intraperitoneally 
with 3 l^t- of sodium acetate labelled in the carboxyl group. 



410 ADVENTURES IN RADIOISOTOPE RESEARCH 

The animals were killed at definite times. 

The same organs in the same group were combined and frozen in 
solid COg. 

A small amount of them were immediately dried at 70° and measured 
as total tissue. 

The ground organs were then extracted with a lioiling mixture of 
ether-alcohol 1/3 for 3 hr. The filtrate obtained was evaporated and the 
residue extracted with petroleum-ether. 

The total fats obtained by evaporation of the petroleum-ether were 
purified following the procedure of Folch and Van Slyke (9). 

The determination of the radioactivity was carried out with a Geiger- 
Miiller counter. The activity of the samples was determined without 
converting these into barium carbonate. 



RESULTS AND DISCUSSION 
a) Liver 

The change in the relative specific activity of the total fat extracted 
from the liver of control mice and mice injected with dinitro-cyclo- 
pentylphenol with time is seen in Fig. 1(a). Each value indicates the 
i*(J content of a fat sample obtained from 5 pooled livers. After admi- 
nistration of labelled acetate, the incorporation of ^*C into fatty com- 
ponents first increases, soon however due to the very rapid turnover rate 
of a liver fatty fraction observed, the labelled fatty molecules will be 
replaced by newly formed ones. As these molecules are formed from 
a much less active medium — the specific activity of the liver acetate 
rapidly decreases with time — they are less active than the degraded or 
emigrated active fatty molecules. Due to these facts, the curve repre- 
senting the change in the specific activity of the liver fat with time soon 
shows a decreasing trend. This is found to be the case already after the 
lapse of 7 — 15 min after the injection of labelled acetate. 

If the rate of fat formation is influenced by the effect of the adminis- 
tered dinitro-cyclo-pentylphenol and accelerated in the first phase of the 
experiments, more ^HJ can be expected to be incorporated into the fat 
of the liver of such animals than in those of controls. The left part of the 
curve will thus show a higher peak in the case of the mice treated with 
the dinitro-compound than in that of the controls. The right part on the 
curve on the other hand can be expected to show a steeper descent for 
the animals injected with the dinitro-compound. tSuch a behaviour is 
actually shown by the curves seen in Fig. 1 and also in those of Fig. 2 in 
which the change of the specific activity of the exhaled CO2 is plotted 
against time, after injection of labelled acetate and succinate. Succinate 



INCORPORATION OF LAB. ACETATE CARBON ("C) INTO TISSUE FRACTIONS 411 



C 

O 
<_) 

2000 



Liver 

Conrrol 

Di nit rocyc I openryl phenol 



t500 - 



000 



500- 




60 mm. 



Fig. 1. Effect of dinitro-cyclo-pentylphenol on the incorporation of 
i*C into total fats (a) and total tissue (b) of the liver of the mouse, 

"C injected as CHgWCOONa. 



is metabolized at a slower rate than acetate and correspondingly in the 
left part of the curve the acetate values are higher, in the right part of the 
curve the succinate values are higher (2). 

A similar trend as indicated by the curves plotted in Fig. la is shown 
by the curves seen in Fig. 16 in which the specific activity of the dry 
liver tissue is plotted as function of time, both for controls and D.P.P. 
injected animals. The ^^C-content of the liver tissue except in a very 
early phase is mainly due to its fatty components but some is still pre- 
sent in acetic acid and other acid soluble tissue components, and to a 
very restricted extent in glycogen and in proteins. That the absolute 
i^C content of the total tissue is much smaller than that of the fatty 
components, is due to the dilution of highly active ftitty acids ])y large 
amounts of slightly active tissue components. 



412 



ADVENTURES IN RADIOISOTOPE RESEARCH 



4,8- 
4,4- 

4,2 
4,0 

o 

j: 3,6 

^3,4^ 

^3,2- 
i 3,0 
°- 2,6- 

i 2.6 
o 

" 2,4-1 
° 2,2- 
2,0 
1.8 



Succinate 




30 



60 



90 120 

Time in minutes 



150 



180 



210 



a40 



Fig. 2. The logarithm of the specific activity of COg excreted is 

plotted against time, following the intraperitoneal injection of iso- 

topic sodium acetate and succinate (Gould et al. (2)). 



b) Skeletal Muscles 

A very different result as stated above is obtained in the study of tiie 
incorporation of i^C into the total dry tissue of skeletal muscles, a large 
share of which is due to the fats present. As seen in Fig. 3, the admi- 
nistration of the dinitro-compound decreases the rate of incorporation 
of 14C into the muscle tissue both in the initial and in the later phases 
of the experiment. Our very restricted knowledge as to fatty acid for- 
mation in the intrusion into the muscles and to that of other labelled 
compounds makes the interpretation of the above results difficult. II 




-f 



---^ DPP. ______: + 



+ 



I 

50 



— r" 
45 



60 mm. 



Fig. 3. Effect of dinitro-cyclo-pentylphenol on the incorporation of 
"C into the total muscle tissue of the mouse, "C injected as 



CHg^COONa. 



IXCOlll'ORATIOX OF LAB. ACETATE CARBON (C) IXTO TISSUE EliACTIOXS 413 

undor the action of the dinitro-compound the formation in or intrusion 
of fatty acids or their precursors into the muscle should be slowed down, 
we would obtain a lower ^*C content in the D.P.P. injected mice. A lower 
J^C incorporation can, however, be tlu> result not only of a depressed 




Brain 
Tolal tissue 



»; 



mm. 




Broin 
Total fats 



mm. 



Fig. 4. Effect of dinitro-cyclo-pentylphenol on the incorporation 
of i*C into total fats (b) and total tissue (a) of the brain of the mouse, 

i^C injected as CHg^COONa. 



formation rate of fats and other labelled components, but also of an en- 
hanced dilution of the labelled acetate or its transportation products by 
endogenous inactive compounds. 

Assuming the half-life time of the average fatty acid molecule in the 
mouse to bel8 days, the metabolism of 1 gm fatty acids present in the 
muscles of a 20 gm mouse will lead to the formation of about 1 millimol en- 
dogenous (at the start inactive, later slightly active) acetic acid. In this 
calculation only the carcass fatty acids are considered (4). Tf under the 
eff(H't of the D.P.P. the carcass fatty acids should be catabolized at 
an enhanced rate, for example, twice as rapidly as in the controls (inac- 
tivation of pyruvate is leading to the accumulation of acetate as well), 
the dilution of the active muscle acetate and its transformation products 



414 



ADVENTURES IN BADIOISOTOi'E RESEARCH 



would be twice as large in the D.P.P. treated animal. If the rate of 
formation of fatty acids would not be influenced in the muscles of such 
a mouse, the incorporation of ^^C into fatty acids would be half only 
in the D.P.P. injected mice of that of controls. Under physiological 




mm. 



Fig. 5. Change of C^* content of brain fractions with time. 



conditions the rate of formation of fatty acids will correspond to their 
rate of degradation. This is no longer the case in the D.P.P. treated 
animal which may rapidly loose some of its fat content. 

It is quite possible that fats or their precursors reach the muscles from 
the liver. Most of the time the i'*C content of the fats in the liver of D.P.P. 
treated mice is lower than the ^*C content of the fats of controls. This 
may also explain the low ^*C values in the muscle fats of the dinitro- 
cyclo-pentylphenol treated mice. Further information on this point 
could be obtained by studying the intrusion into the muscles of labelled 
fatty acids, labelled aceto-acetonate or other labelled precursors intro- 
duced into the circulation. 



INCOKPOKATIOX OF LAB. ACETATE CAllBOM ("C) INTO TISSIK FRACTIONS 4] 5 



c) Brain 

The results plotted in Fig. 4(a) and 4(&) indicate a slight effect only 
of injection of D. P. P. on the metabolic steps of the brain in which acetate 
carbon participates. The total labelled acetic acid conient of the ra1 



Skin 

Total tissue 




60 mm. 



Heart 
Total tissue 




60 min. 



Fig. 6. Effect of dinitro-cyolo-pentylphenol on the im^orporation 
of i*C into total tissue of skin (a) and heart (b) of the mouse, ^*C 

injected as CHgi^COONa. 



was found by Gould et al. (2) to decline during the same time- 
interval to 1/3 of its initial value as well. We can expect a very rapid 
metabolic rate in the brain in view of the high oxygen consumption of 
that organ. The striking decline in the activity of the fat free brain 
with time is seen in Fig. 5. After 30 min only, the activity of the acid, 
soluble and protein fractions is less than half of the value observed after 
10 min. The slight increase in the ^^C content demonstrated by the last 



416 ADVENTURES I\ RADIOISOTOPE RESEARCH 

part of the upper curve is presumably due to an increase in the activity 
of the protein fraction, which as shown by the lower curve is increasing 
with time. 

d) Skin and Heart Muscle 

For the skin and heart muscle we determined the effect of DP. P. 
injection on i^Q incorporation into the total tissue only. The curves 
plotted in Fig. 6 (a) representing the results obtained for the skin, show a 
similar trend to the corresponding curves obtained for the skeletal muscle. 

The first part of the curves which show ^^C incorporation into the 
heart tissue 6(&), indicates an accelerated rate of i^C incorporation into 
the total organ under the effect of D.P.P. administration. The further 
trend of the figures is quite complicated, which may be due at least 
partly to the fact that we deal with the total tissue which contains fat 
and acid soluble fractions of appreciable i^c content and less active 
protein and glycogen fractions. 

The trend of the curve denoting the change in the specific activity of 
the heart muscle tissue under the effect of D.P.P. markedly differs 
from that of the curves obtained for the skeletal muscle. This difference 
suggests a specific effect of D.P.P. on the heart. 

Summary 

The rate of incorporation of i*C after injection of earboxyl labelled acetate 
in the liver, brain, skin, skeletal and heart muscle of control mice is compared 
with the values obtained with organs of mice to which dinitro-cyclo-pentylphenol 
(D.P.P.) was administered. 

Administration of D.P.P. increases the rate of incorporation of i*C into hver 
fat and total liver tissue in the first 10 minutes of the experiment and decreases 
in the later part. Such a behaviour is expected if D.P.P. increases the rate of ace- 
tate metabohsm in the liver. 

Administration of D.P.P. has a slight effect only on i^C incorporation into 
brain fat or total brain tissue. 

The D.P.P. injected mice take up less "C in the skeletal muscle tissue and 
skin tissue than do the controls. 

References 

IK. Block and D. Rittenberg, J. Biol. Chem. 159, 45 (1945). 
2R. G. Gould, F. M. Sinex, I. N. Rosenberg, A. K. Solomon and A. B. 
Hastings, J. Biol. Chem. 177, 197 (1949). 

3 A. PiHL, K. Bloch and H. S. Anker, J. Biol. Chem. 183, 441 (1950). 
4G. Hevesy, Nature 164, 1007 (1949). 

5 G. Hevesy, R. Ruyssen and M. L. Beeckmans, Experientia in print. 

6 C. Heymans and H. Casier, Arch. int. Pharmacodyn. 50, 20 (1935). 

' B. Terada and M. L. Tainter, /. Pharmacol, and exp. Therap. 54, 454 (1935). 

8 W. F. LoOMis and F. Lipmann, J. Biol. Chem. 173, 807 (1948): 179, 503 (1949). 

9 J. FoLGH and D. D. Van Slijke, Proc. Soc. Exp. Biol. Med. 41, 514 (1939). 



Originally communicated in Exp. Cell Res. 3, lid (1952) 

41. DETERMINATION OF THE RATE OF RENEWAL 
FROM THE RATE OF DISAPPEARANCE OF LABELLED 

MOLECULES 

George Hevesy 

From the Institute for Research in Organic Chemistry, 
University of Stockholm 

The rate of renewal of a type of molecules is usually calculated from the 
rate of incorporation of the labelled atoms of the pertinent precursor 
in the molecules considered. If we wish, for example, to known the 
percentage of desoxyribonucleic acid molecules of the rat spleen, which 
are formed in the course of two hours, we administer labelled sodium 
phosphate to a rat and two hours later we compare the specific activities 
of the desoxyribonucleic acid P, and inorganic P extracted from the 
.spleen. If the ratio of these specific activities is found to be 0.02, the 
specific activity of the inorganic P remained constant during the experi- 
ment, and this P can be considered to be the pertinent precursor of 
desoxyribonucleic acid P, we can conclude that in the tumour 2 per cent 
of the desoxyribonucleic acid molecules are present which were formed 
during the experiment or, more correctly, that at least 2 per cent of 
these molecules were formed in the course of two hours. If it takes some- 
time that the labelled precursor reaches the site of desoxyribonucleic 
acid syntesis the first phase of the synthesis of this compound will not 
be indicated by the tracer and we shall correspondingly underestimate 
its rate of synthesis formation. 

If w^e protract the experiment, the specific activity of the inorganic 
!•* of the spleen decreases more and more and this decrease is followed 
by a decrease in the specific activity of desoxyribonucleic acid P. We can 
also calculate the rate of renewal by comparing the specific activities of 
desoxyribonucleic acid P and inorganic P in this declining activity phase 
of the experiment. 

The rate of loss of ^"^P by desoxyribonucleic acid molecules in the late 
phase of the experiment is independent of the precursor problem, but 
parallel with a loss ^^p by strongly active "old" desoxyribonucleic acid 
molecules the formation of less active "new" molecules takes place 
for example in the spleen and the rate of incorporation of ^-P in these 
molecules is partly determined by the specific activity of the pertinent 
precursor. Thus by replacing the calculation of the renewal rate from 

27 Heresy 



418 ADVENTURES IN RADIOISOTOPE RESEARCH 

increasing values of the specific activities of desoxyribonucleic acid 
with time by a calculation based on decreasing specific activity values 
with time, we cannot fully eliminate the difficulty arising from the lack of 
knowledge of the pertinent precursor of desoxyribonucleic acid P. We 
meet, however, very different conditions when faced with the task to 
calculate the rate of renewal of a carbon labelled compound from specific 
activity or similar data. 

i^C of rapidly metaboUzed carbon compounds such as acetate, glucose 
and so on, is within a comparatively short time exhaled to a very large 
extent as carbon dioxide. Correspondingly the rate oflossof i*C by fatty 
acid molecules in a later phase of the experiment in which, for example, 
labelled acetate was administered to the rat, is no longer a resultant of the 
disappearance of "old" strongly labelled molecules and the formation 
of "new" less strongly labelled ones but, at least in the first approxima- 
tion, the result of the decay of labelled molecules only. By following the 
rate of decrease of the i^c content of fatty acids extracted from the 
organs of the rat we can thus calculate the renewal rate of fatty acid 
molecules in an analogous way to that, in which we calculate the period 
of decay of a radioactive body from its "decay curve." In the present 
note the calculation of the renewal rate of the fatty acids of the liver 
from the rate of decrease of the i^C content of the fatty acids in the 
rat injected with acetate labelled in the carboxyl group is described. 



EXPERIMENTAL 

44 rats weighing 190 to 244 gm were injected intraperitoneally each 
with 0.2 ml of phys. sodium chloride solution containing acetate labelled 
in the carboxyl group of 10—26 fi C activity. The animals were killed by 
decapitation and the total fat, fatty acids, neutral and phosphatide fatty 
acids, and also cholesterol of the liver, secured as described previously. 
(2, 8) The activity of the samples was compared without conversion into 
barium carbonate. 



RESULTS 

In an early investigation, using deuterium as an indicator, Stetten" 
and Boxer (12) found the half life-time of fatty acids of the rat liver 
to amount to 1.9 days. Rittenberg and Block, (11) feeding ^^C label- 
led acetate, observed a more rapid incorporation of ^^C into saturated 
than into unsaturated fatty acids of the mouse hver. In a more recent 
work, PiHL et at. (8, 9) compared the ^^C content of the fatty acid carbon 
(at the end of the experiment taking many days) with the average value 



THE EATE OF RENEWAL I'JUJ.M KATE OF DISAPrEAKAXCE OF LAii. .MOLECULES 419 

of the i^C content of the precursor carbon prev aihng during the experi- 
ment. The saturated fatty acids of the liver were found to reach half of 
their maximum isotope concentration in less than one day, the unsatu- 
rated acids in about two days. 

When investigating the effect of muscular exercise on the incorporation 
of i^C into liver fats, the present writer (5) found after the lapse of 4I/2 hr 
the i^C content to be only one third of that detected 20 min after injecting 
labelled acetate into the mouse; however, according to the above men- 
tioned data, in the course of two hr. the loss of i*C by liver fatty acids 
should be less than 10 per cent. Obviously, a fatty fraction is present in the 
liver which has a much shorter half-life time than one day. Ruyssen, 
Beeckmans and the author (6) investigated the renewal rate of fatty 
acids in the liver of the mouse shortly after administration of labelled 
acetate. They obtained the result that the i*C content of the liver fatty 
acids increase rapidly during about the first 30 minutes, this increase 
being followed by a rapid decrease. Furthermore, Beeckmans and 
Elliott (2) could demonstrate that in both the saturated and unsatura- 
ted fatty acid fractions this early increase is followed by a rapid decline 
in the ^^C content. The liver must thus contain one or more rapidly 
renewed fatty acid fractions. To discard the effect of this rapidly meta- 
bolizing fatty acid fraction we studied the decrease in the ^^C content 
of liver fatty acids one or 1 | days after administration of labelled 
acetate only and followed it till the lapse of 4 or 4 | days. As we in our 
determination of the renewal rate disregard those labelled fatty acid 
molecules which are formed during the experiment we have obviously 
quite apart from the above considerations to wait for about 1 day after 
injecting labelled acetate before securing the samples. 

Gould et al. (4) found that after the lapse of three hr. only 85 per cent 
of the acetate ^^C injected into the rat is already exhaled as ^^COg. In the 
later phase of the experiment the rapid loss of the exogenous acetate 
i*C may to some extent be compensated by formation of labelled endo- 
genous acetate. Pihl et al. (9) kept the activity of body acetate of the 
rat at a constant level by feeding labelled acetate; in these experiments 
an increase in the body acetate activity could be observed after the lapse 
of ten days which was due to the formation of endogenous acetate of 
appreciable C^* content. Under our experimental conditions (injection 
of the labelled acetate at the start of the experiment) the activity of 
endogenous acetate formed during the experiment was, however, negli- 
gible. 

In Fig. 1 the decrease in the specific activity of fatty acid carbon 
with time l| f o 4 ^ days after injection of labelled acetate is plotted; 
also data obtained by Pihl et al. (9) for the increase in the percentage 
of labelled fatty acids with time are seen. The figure contains thus 
both "rise curves" and "decay curves". From the data of Pihl et al. 

27* 



420 



ADVENTURES IN RADIOISOTOPE RESEARCH 



follow that the half life-time of saturated fatty acids is less than one 
day and that of unsaturated fatty acids about two days ; our data 
indicate practically the same result, 0.8 and 2.2 days, respectively. 
A closer coincidence is hardly to be expected in view of the fact that 
even when comparing fatty acid turnover in the liver of rats of the 
same race, age and weight, very appreciable fluctuations appear. Ame- 



iOO 



50- 



25- 






icorporation of ac'efate "Cinto saturorecl 
faffy acids 



'>..^oss of "c by saturated fatty acids 



-V 



Fig. 1. Rate of incorpo- 
ration of acetate C^* 
(PiHL et al.) and rate of 
loss of 1*0 by saturated 
fatty acids extracted from 
rat liver. ("Rise curve" 
and "Decay curve".) 



4 days 



100 



ncorporation of acetate '^C into 
unsarurated fatty acids 




Less of '*Cby unsaturated fatty acids 



4 days 



Fig. 2. Rate of incorpo- 
ration of acetate "C 
(PiHL et al.) and rate of 
loss of "C by unsaturated 
fatty acids extracted from 
rat liver. 



lioration of purification or measuring methods would hardly lead to more 
accurate mean renewal times, such could be obtained only by investi- 
gating an appreciably larger number of animals. 

The significance of the data obtained is restricted, as both the satu- 
rated and unsaturated fatty acids represent a mixture of components 
having different turnover rates, some components of the unsaturated 
fatty acid mixture as linoleic or linoleic acid are not synthesized in the 
animal organism, and thus are not labelled. While Pihl and Bloch [8] 
state that the linoleic acid content of the rat liver is almost negligable, 
these authors find neutral fatty acids to contain 16 per cent, phospha- 
tide fatty acids and 8 per cent of linoleic acid. From the fatty acids ex- 
tracted from the liver of rabbits Popjak and Beeckmans [10] found 
that when the acetate was given to the animals for 20 hr the specific 



THE RATE OF RENEWAL FROM RATE OF DISAPPEARANCE OF LAB. .MOLECULES 421 

activity of phosphatide fatty acids was larger than that of the glyceride 
fatty acids, in experiments of longer duration, however, no significant 
difference in the ^K' content of th(> two l^^pes of fatty acids could be 
observed. 

In all our experiments 1 to 4 days after injecting the labelled acetate 
the specific activity of the total fatty acids of neutral fat of the rat liver 
was found to be a few per cent higher than the specific activity of phos- 
phatide fatty acids, while according to Pihl and Block (8) 3 days 
after feeding labelled acetate to rats kept on lipid free diet, the phos- 
phatide fatty acids are 6 to 13 per cent more active than the total fatty 
acids of neutral fat. 

As mentioned above, investigation of the incorporation of i*C into 
fatty acids of the liver shortly after the administration of labelled acetate 
to mice revealed the existence of a rapidly metabolising fatty acid fraction. 
A rapid decrease in the ^^C content of total fat, neutral and phosphatide 
fatty acids of the liver of the rat is also observed shortly after the ad- 
ministration of labelled acetate but is less pronounced than in the case 
of the mouse liver. Some results obtained when injecting several rats 
each of which being killed at a different time after intraperitoneal 
injection of the labelled acetate are shown in Tables la and b. 

These and similar results are to be interpreted cautiously, among 
others because — in contrast to the experiments on mice where pooled 
livers of a large number of mice where extracted — in the present inves- 
tigation each point indicates the result obtained when extracting a single 
liver only. The fatty acid metabolism may strongly vary from animal to 



Table 1. — Percentage injected acetate 
i''C present in 1 mgm of neutral total 

FATTY ACIDS OF NEUTRAL FAT EXTRACTED FROM 

THE livp:r of 182 — 190 gm rats 



Time after injection 



I'erceiitage injected x 10^ in 
1 mgm total fatt}- acids 
of neutral fat 



20 mill 


a) 
b) 


37 5 


80 ,, 


51 4 


110 „ 


fi3 


140 „ 


21 1 


170 „ 


23 2 


240 „ 


•'8 3 


20 „ 


.56 "> 


.-)0 ,, 


66 


SO , 


79 2 


110 „ 


38 







422 ADVENTURES IN RADIOISOTOPE RESEARCH 

animal, even when experimenting with animals of the same race, age and 
weight. The reason of these variations is inherent partly in the constitu- 
tion of the livers compared, and is presumably partly the result of a 
difference in the amount and time of food taken in by the rat. A diffe- 
rence of minutes only in the time when the animal last took in food may 
influence the renewal rate of the fatty acids of the liver. 

In spite of the great variations in the rate of i^C incorporation into 
liver fatty acids the results shown in Table 1 and further similar results 
indicate a more rapid loss of fatty acid i*C in the first hours of the ex- 
periment than in the later phase, the maximum ^^C incorporation being 
observed 1 — 2 hr after injecting the acetate. One may be inclined to 
interpret the rapid decrease in the ^^C content of the fatty acids during 
the first hours of the experiment as a consequence of the presence of 
an impurity in the fatty acid fraction. After purification of the fatty 
acids or the phosphatides according to Folch and van Slyke's method 
(3) the decrease was still observable. For the fatty acids of the mouse 
liver the i^C content of the isolated lead salts has furthermore shown 
a similar time dependency to that of fatty acid mixture (2). Lehninger's 
copper-calcium method of purification, while increasing the activity 
figures with 10—20 per cent through removal of less active fractions, 
did not influence significantly the time dependency of activity figures 
obtained. The same applies to the already mentioned purification with 
colloidal iron hydroxyde. The specific activity of phosphatide fatty 
acids, for example, purified by this method, was increased up to 40 
per cent ; however, the percentage change in i^C content of the fatty 
acids secured at different times after injecting labelled acetate to the 
rat was not significantly influenced by the purification process. 



Summary 

Since most of the i*C injected as acetate into the rat is exhaled within a day, 
incorporation of "C into fatty acids of the hver after that date can be neglected 
in the first approximation. From the rate of loss of "C by the fatty acids of the 
hver of rats, to which one or more days previously labelled acetate was admi- 
nistered, the rate of renewal of the fatty acids can thus be calculated. The half- 
hfe of the saturated fatty acid mixture was found to be 0.8 days, that of the unsa- 
turated fatty acid mixture (1 to 4 days after injection of the labelled acetate) 
to amount to 2.2 days. 



THE KATE OF RENEWAL FROM RATE OF DISAPPEARANCE OF LAB. MOLECULES 423 

References 

1. R. Abbams and J. M. Goldtnger, Arch. Biochcm. 30, 2(jl (1951). 

2. I. M. Beeckmans and G. Elliot, Nature, 167, 200 (1951). 

3. J. FoLCH and D. D. Van Slyke, Proc. Soc. Exptl. Biol. Med. 41, 514 
(1939). 

4. R. G. GoTJLD, F. M. SiNEX, I. N. Rosenberg, A. K. Solomon and A. B. 
Hastings, J. Biol. Chem. 177, 295 (1949). 

5. G. He^tesy, Nature 164, 1007 (1949). 

6. G. Hevesy, R. Ruyssen and L. M. Beeckmans, Experimentia 7, 144 
(1951). 

7. J. Ottesen, Personal communication. 

8. A. PiHL and K. Block, J. Biol. Chem. 183, 431 (1950). 

9. A. PiHL, K. Bloch and H. S. Anker, J. Biol. Chem. 183, 441 (1950). 

10. G. PopjAK and M. L. Beeckmans, Biochem. J. 47, 233 (1950). 

11. D. Rittenberg and K. Bloch, /. Biol. Chem. 160, 417 (1945). 

12. D. Stetten Jr. and G. E. Boxer, J. Biol. Chem. 155, 231 (1944). 



424 ADVENTURES IN RADIOISOTOPE RESEARCH 



Comment on papers 41 — 43 

In some of their earliest classical investigations, Sc hoe nheimer and Rittenberg 
using deuterium as an indicator found the half-hfe of fatty acids of the liver of 
the rat to be from 1 to 2 days. Investigating with Dreyfus the effect of irradiation 
on the turnover rate of the fatty acids of the liver of mice very shortly after admi- 
nistration of "C labelled acetate (paper 81), we found the presence in the Uver 
of a fatty acid fraction having the half- life of some minutes only. No such fraction 
was found in the fatty acids extracted from the brain or the muscles (paper 39). 
Beeckmans and Elliot (1951) succeeded in our laboratory in demonstrating 
that both the saturated and unsaturated fatty acids of the liver contain a short - 
living fraction, and Swan (1951) could show that we are not faced with only 
a re-carboxylation of the molecule, but with a renewal of all its carbon atoms. 
Recently, a short-living fatty acid fraction was found also in the liver of the 
lat (TovE 1956). 

The half-life of molecules is usually determined by following the incorporation 
of the tracer into the newly formed molecules. As described in paper 41 we can 
also determine the half-life of the liver fatty acids by labelling these and following 
the rate of decrease in their activity. 



References 

L. M. Beeckmans and G. Elliot (1951) Nature 167, 200. 

G. A. Swan (1951) Ark. Kemi 3, 167. 

S. B. TovE, J. S. Andrews and H. C Lucas (1956) J. Biol. Chem. 218, 275. 



Oi'i^inally publishod in Acta rhijaiol. Scand. 1, 347 (1!)41) 

42. RATE OF PENETRATION OF IONS THROUGH THE 

CAPILLARY WALL 

L. Hahn and G. Hevesy 

From the Institute of Theoretical Physics, University of Copenhagen 

In this paper, the results of experiments are communicated which were 
carried out in order to get information on the rate of passage of the ions 
of important constituents of the plasma as sodium, potassium, chlorine, 
and phosphate through the capillary wall. Crystalline substances intro- 
duced into the circulation will soon invade the extracellular fluid of the 
body. On this fact is based the method usually applied to determine 
the size of the extracellular space. Sucrose, sulphocyanate, or sulphate 
introduced into the human circulation were found (Lavietes et al., 
1936), for example, to be completely distributed between the plasma 
and the tissue space in the course of two or three hours. A complete 
distribution of thiocyanate in the extracellular space of rabbits in the 
course of half an hour is recorded (Krogh, 1937). 

The partition of a substance introduced into the circulation between 
plasma and the extracellular fluid involves two processes : (1) penet- 
ration across the capillary wall and (2) distribution by fliffusion and 
convective processes in the capillary and the extracellular fluids. The 
intrusion into the capillaries will play a secondary role, only, in view of 
the very short distances between the capillaries. Taking the length of the 
distances involved (Krogh, 1926) to be less than 60 ju and the diffusion 
coefficient of the substance investigated to be at least 1 cm^ per day, the 
time necessary to displace, for example, a sodium ion from one end of 
the capillary space to the other, or from one end of the corresponding 
extracellular space to the other, will be less than 2 sec^^). We arrive 
at this result by considering the propagation by diffusion only of the 
substance which penetrated the capillary wall. The fluid is, however, 
not without a circulation of its own, and this circulation will possibly 
shorten the time arrived at in the above calculation. 

By introducing some sodium chloride into the circulation and by 
measuring the time it takes for a certain fraction to leave the circulation, 

(i^Thc mean displacement of a particle T= |/^2 D, where D is the diffusion 
coefficient . 



426 ADVENTURES IX RADIOISOTOPE RESEARCH 

it should be possible to measure the rate of passage of sodium chloride 
through the endothelium. However, when carrying out these experi- 
ments we meet the following difficulties : (a) Not only does the cir- 
culation get rid of excess sodium chloride by giving off salt to the extra- 
cellular space, but also by taking water up from the tissues. Keyes 
(1937) found, when studying the fate of sucrose intravenously injected 
into man, that osmotic equilibrium by a shift of water takes place from 
three to ten times as fast as sucrose exchange. The rate of disappearance 
of the excess sodium chloride will, thus, not measure the rate of passage 
of sodium chloride through the capillary wall but a more complex 
process, (b). The resistance of the endothelium to the passage of 
sodium and chloride may be quite different, (c) The introduction 
of appreciable amounts of sodium chloride into the circulation 
will disturb the normal conditions prevailing in the circulation. When 
one tries to eliminate this difficulty by introducing small amounts 
only, the analytical difficulties become almost unsurmountable. All these 
difficulties can be eliminated by injecting into the veins labelled sodium 
chloride (sodium chloride containing some radioactive ^^Na of negligible 
weight) and by measuring the rate of disappearance of the active ions 
from the plasma, i. e. the decrease of the radioactivity of the plasma. 
We are not determining in these experiments the rate of influx of excess 
sodium chloride from the plasma into the extracellular fluid but the rate 
of exchange between labelled plasma sodium and non-labelled extra- 
cellular sodium, as the number of sodium (^^Na -j-^^Na) atoms of the 
plasma remains practically constant all through the experiment. The 
rate of exchange will be determined by the permeability of the capillary 
wall to sodium ions and will, thus, be a measure of this permeability. 
We carried out also experiments with radio-potassium, radio -chlorine, 
radio-bromine, and radio-phosphate, while heavy water was used as an 
indicator for water in the study of permeability of the endothelium to 
water. The measurement of the distribution of radio-sodium between 
plasma and the extravascular space of the rabbit was previously used 
to determine the extracellular A'olume of the rabbit (Griffith and 
Margraith, 1939 ; Hahts- et al., 1939). 



EXPERIMENTAL PROCEDURE 

Radioactive sodium and potassium were prepared by bombarding NaOH and 
KOH, respectively, with high speed (10 million volts) deuterons. The hydroxydes 
were neutralized with hydrochloric acid and the solution thus obtained was injected. 
Radioactive chlorine and broniine were prepared by bombarding NaCl and NaBr, 
respectively, with deuterons. The active chlorine and bromine obtained were 
driven off as HCl and HBr, respectively, and were collected in a sodium hydroxyde 
solution. This procedure was chosen to get rid of the active sodium simultaneously 



RATE OF PENETKATION OF IONS THROUGH THE CAPILLARY WALL 



427 



produced with the active halogens. We are much indebted to Dr. J. C. Jacobsen 
and Mr. O. N. Lassen for preparing the radioactive substances by making use 
of the Copenhagen cyclotron. 

About 3 cc. solution containing the radioactive substances of an activity of 
about 1 microcurie was applied. The salt concentration of these solutions was 




3 4 

Min. 



Fig. 1. Rate of disappearance of various labelled ions from the plasma. 



brought up to a physiological level by adding non-active sodium chloride. The 
solution was injected into the jugularis of the rabbit and blood samples of about 
1 cc. were collected at intervals from the carotis. Plasma samples of known weight 
were dried and their radioactivity was compared by using a Geiger counter. For 
comparison of the radioactivity of plasma and muscle samples the samples were 
ashed at about 400° and the plasma ash mixed with non-active muscle ash of the 
same weight as the corresponding active muscle ash sample. Blood and muscle 
samples were secured simultaneously from the narcotized rabbit. 



RESULTS 



The results obtained are seen in Tables 1 to 5 and Figs. 1 to 3. The 
tables contain data on the percentage of the labelled element injected 
still present in 1 cc. plasma at various intervals. The volume of diluting 
lluid necessary to bring down the concentration of the substance in- 



428 



ADVENTURES IN RADIOISOTOPE RESEARCH 



jected to that found after a given time is also stated. Furthermore, the 
diluting volume is expressed in percent of the rabbit's body weight . 
We shall first compare the rate of disappearance of sodium, chlorine, 
and bromine from the circulation. This comparison encounters no 
difficulties since practically the sole outlet of these elements from 
the circulation is the extracellular body fluid, though some ^^Na is 
taken up by the bone apatite (Hahn et ah 1939). No great diffe- 




FiG. 2. Rate of disappearance of various labelled ions from the plasma. 

rence is found between the rate of passage of sodium, chlorine, and 
bromine through the capillary wall but the values obtained for dif- 
ferent rabbits show fairly large variations. These variations are to some 
extent due to differences in the size of the extracellular space. 

A comparison of the rate of passage of potassium, phosphate, and 
water with that of sodium, chlorine, and bromine encounters some 
difficulties since potassium, and the same applies to phosphate and 
water, has an additional outlet into the tissue cells in contrast to the 
first mentioned group. The amount of ^-K lost by the blood after the 
lapse of a given time is the resultant of the amount penetrated into 
the tissue fluids and that returned from the latter into the blood. When 



1 In experiments taking up to 1 hour, the amount of ^^Na lost by excretion 
is less than 1 per cent of the amount administered. 



BATE OF PENETRATION OF IONS THKOUGH THE CAPILLARY WALL 



429 



Tablk. 1. — U.vrE OF Di.sAPPK.\RANCK OF ^sci From thk Circu- 
LATiox OF Rahbits Wkiohing 2.5 AXD 2.4 KOM, Rkspkctivkly 



• 


IVri'oiit ot "01 injected present 
in 1 gm jilnsmri 


Diluting fluid volume 


Time in uiiii 


in cc. (apparent 

extracellular 

volume) 


in percent of 
liody weight 




Rahhit I. 




0.37 


0.622 


161 


6.4 


0.73 


0.486 


206 


8.2 


l.OI 


0.475 


211 


8.5 


1.48 


0.408 


245 


9.8 


2.05 


0.400 


250 


10.0 


3.8 


0.329 


304 


12.2 


10.5 


0.224 


446 


17.8 


18.5 


0.188 


532 


21.3 


35 


0.182 


550 


22.0 




Rabbit II. 




0.3 


0.62 


161 


6.7 


0.9 


0.28 


357 


14.9 


2.5 


0.174 


575 


24.0 


4.7 


0.165 


607 


25.3 


8.3 


0.161 


622 


26.0 


16.5 


0.150 


668 


27.8 


26 


0.143 


700 


29.2 


51 


0.128 


783 


32.6 



licsides the interspaces the cellular space opens an outlet to the '^^K 
leaving the circulation, the amount returning from the tissue fluids 
into the hlood ^vill be reduced and, thus, the resultant ^-Iv concentration 
of the plasma will be lowered. Though the potassium content of the 
cells is only partly replaced by ^^K during the experiment in view of 

Table 2. — Rate of the Disappeakaxce of ^^Br from the 

CmCTTLATION OF A RaBBIT WEIGHING 2.7 KGM 



TiirK; in ni 



I'crcent ot '"Hr injectefl present 
in 1 "^m plasma 



Diluting fluid volume 



in ce. 
(apparent extra- 
cellular volume) 



in percent of 
bodv weight 



1.0 


0.37 


270 


10.0 


2.2 


0.27 


370 


13.7 


8.1 


0.21 


475 


17.6 


16.3 


0.18 


556 


20.6 


32 


0.14 


715 


26.5 


58.5 


0.12 


835 


30.9 



430 



ADVENTURES IN RADIOISOTOPE RESEARCH 



the low potassium content of the plasma and the high content of the 
tissue cells, the additional outlet opened by the intrusion of ^^K into 
the cells in experiments taking one hour, makes out about five times the 
normal outlet of intrusion of these ions into the interspaces. 

The total water content of the cells can be entirely replaced by labelled 
water. Since the volume of the cellular body water is about twice as 
large as that of the extracellular fluid through the intrusion of labelled 
water into the cells a substantial additional outlet of the labelled water 
molecules of the plasma is